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Repellancy, Lethal Time, and Transference of Residual Insecticides Used for Pharaoh Ant Control


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REPELLENCY, LETHAL TIME, AND TRANSFERENCE OF RESIDUAL INSECTICIDES USED FOR PHARAOH ANT CONTROL By DAVID ANTHONY MELIUS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by David Anthony Melius

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This thesis is dedicated to my mom, Donna Melius.

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iv ACKNOWLEDGMENTS I would like to thank my parents and sister s for their continuous support. I would like to thank my friends including Colin Hick ey, Justin Saunders, and Ryan Welch. I greatly appreciate the efforts of Tiny Willis and Gil Marshall in helping to gather supplies for all of my experiments, as well as for their constant help and generosity. I also would like to thank Paul Kaiser for his help in setting up, recording data, and taking down experiments. I would like to thank Debbi e Hall and Josh Crews for their consistent problem solving skills. I would also like to thank Nancy Sanders for her kindness, support, and encouragement. I greatly appreciate the effo rts of Jane Medley in preparing extension poster and booklet publications with me and other students in the urban lab. I would like to thank my advisor, Dr. Ph il Koehler, for both his financial support as well as continual help with statistics and experimental design. He has also provided many opportunities for developing presenta tions. These involved many different audiences including pest cont rol operators, pesticide comp any technicians, master gardeners, and other entomologi sts. These presentations have greatly helped me hone my knowledge on urban pests and entomology. I greatly appreciate Dr. Richard Patte rson, who has always been extremely accommodating. I would also like to thank Dr David Williams for his constant help and advice. He has always been available as soon as I need help. He has a vast knowledge of ant biology, control, and experimentation that he was always willing to share with me.

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v Finally, I would like to thank my girlfrie nd, Kendra Pesko. I greatly appreciate her advice and editorial skills. She has help ed me get through both good times as well as bad.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 Origin and Distribution.................................................................................................4 Food and Foraging........................................................................................................6 Medical Importance......................................................................................................7 Control........................................................................................................................ ..8 3 MATERIALS AND METHODS...............................................................................13 Insects........................................................................................................................ .13 Insecticide Treatments................................................................................................13 Repellency Assay........................................................................................................14 Lethal Time Assay......................................................................................................14 Bridge Assay...............................................................................................................15 Data Analysis..............................................................................................................16 4 RESULTS...................................................................................................................20 Repellency Assay........................................................................................................20 Lethal Time Assay......................................................................................................20 Bridge Assay...............................................................................................................21 5 DISCUSSION.............................................................................................................37 Foreword.....................................................................................................................37 Repellency Assay........................................................................................................38 Lethal Time Assay......................................................................................................40

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vii Bridge Assay...............................................................................................................41 Summary.....................................................................................................................44 APPENDIX MANAGEMENT OF INSECT COLONIES...............................................45 LIST OF REFERENCES...................................................................................................47 BIOGRAPHICAL SKETCH.............................................................................................52

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viii LIST OF TABLES Table page 4-1 Distribution of 50 worker ants in release, untreate d, and water, fipronil, or lambda-cyhalothrin treated Petri dishes...................................................................24 4-2 Repellency of fipronil or lambda-c yhalothrin treated glass to pharaoh ant workers.....................................................................................................................25 4-3 KD values of pharaoh ant workers when exposed to glass or wood treated with fipronil or lambda-cyhalothrin.................................................................................26 4-4 LT values of pharaoh ant workers and queens when exposed to glass or wood treated with fipronil or lambda-cyhalothrin.............................................................27 4-5 Percent mortality of pharaoh ant workers in response to placement of glass or wood bridges treated with water, fipronil, or lambda-cyhalothrin...........................28 4-6 Percent mortality of pharaoh ant queens in response to placement of glass or wood bridges treated with water, fipronil, or lambda-cyhalothrin...........................29 4-7 Mass of brood before and after placem ent of bridges treated with fipronil or lambda-cyhalothrin...................................................................................................30

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ix LIST OF FIGURES Figure page 3-1 Repellency assay setup showing locati on of release, treat ed, and untreated dishes........................................................................................................................1 7 3-2 Bridge assay setup showing location of nest cell, bridge, and food sources............18 3-3 Glass bridge assay setup show ing ants trailing along bridge...................................19 4-1 Series of photographs of Control (r eplicate 1) showing nest cell over time............31 4-2 Series of photographs of Control (r eplicate 2) showing nest cell over time............32 4-3 Series of photographs of lambda-cyhalo thrin (replicate 1) showing the nest cell over time...................................................................................................................33 4-4 Series of photographs of lambda-cyhalo thrin (replicate 2) sh owing nest cell over time........................................................................................................................... 34 4-5 Series of photographs of fipronil (re plicate 4) showing nest cell over time............35 4-6 Series of photographs of fipronil (re plicate 5) showing nest cell over time............36

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science REPELLENCY, LETHAL TIME, AND TRANSFERENCE OF RESIDUAL INSECTICIDES USED FOR PHARAOH ANT CONTROL By David Anthony Melius December 2005 Chair: Philip Koehler Major Department: Entomology and Nematology Pharaoh ants, Monomorium pharaonis (L.), are nuisance pests, which have polygynous, polydomous colonies, and repr oduce by budding, making control very difficult. Baiting is generally believed to be the only control method that results in pharaoh ant colony elimination. Effective bait s should not be repellent, possess a slow acting toxicant, and be readily transferred between ants. If a residual treatment could mimic the characteristics of an effective bait toxicant, it c ould be an effective treatment for pharaoh ants. Fipronil and lambda-cyhalothri n were evaluated as residual treatments for infestations of the pharaoh ant. Both fipronil and lambda-cyhalothrin eff ectively killed pharaoh ant workers and queens when they were exposed to the treatmen t. No degree of repellency was found for either insecticide. Glass and wood panels were used to estimate knock down and lethal time values of pharaoh ant workers and queen s. Lambda-cyhalothr in exhibited low LT50 values for workers on both glass and wood surfaces tested (LT50 values = 137.84 and

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xi 158.24 min for glass and wood respectively) indicating that foraging pharaoh ant workers would have very little time to return to the nest. Fipronil treated glass also resulted in a fairly low lethal time value (LT50 = 496.54 min). A delay in mortality >24 h for pharaoh ant workers was only evident in the fipronil treated wood group (LT50 = 2396.00 min), indicating that foraging workers w ould have time to return to the nest. Similar results were found for pharaoh an t queens exposed to fipronil and lambdacyhalothrin. A bridge experiment was conducted in order to determine if a residual treatment of fipronil or lambda-cyhalothrin could be effectively transferred from foraging workers to affect the rest of the colony. Glass treatme nts for both insecticides were effective in eliminating ~80% of the worker populati on. However, no significant queen mortality was evident throughout the test. Wood bridges treated with lambda-cyhalothrin appeared to lose effectiveness after 2 wk and only resu lted in 63% worker ant mortality after 21 d. Fipronil treated wood was the most effectiv e treatment, with 100% worker mortality, 80% queen mortality, and a heavy reduction in brood after 21 d. A delay in mortality >24 h, and preferably in the range of 3-5 days was crucial for effective transference of inse cticides throughout the colony. This was only evident in wood treated with fipronil. Some of the colonies in the fipronil applied to wood treatment were eliminated. Fipronil residual treatments applied to absorbent surfaces, such as wood, showed the greatest potential fo r management of pharaoh ant infestations.

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1 CHAPTER 1 INTRODUCTION Pharaoh ants, Monomorium pharaonis (L.), are nuisance pests, which have polygynous, polydomous colonies, and genera te new colonies by budding. Budding involves the carrying of brood by workers to a new nesting location. Queens are not necessary for the formation of a new satellite nesting location but will sometimes accompany the movement (Edwards 1986). An ts often share res ources between these satellite nests (Holldobler and Wilson 1990) This budding behavior has aided the pharaoh ant in becoming an intense household pest. The slightest disturbance, including spraying repellent insecticides, can lead to th e formation of new satellite nests, making it very difficult for pest control operators to manage pharaoh ant infestations (Holldobler and Wilson 1990, Buczkowski et al. 2005). Colonies typically employ only a small percen tage of workers to serve as foragers (Williams 1990, Vail 1996). This behavior also makes management extremely difficult. Control measures must also include methods of eliminating the repr oductive caste of the colonies. Direct treatment of all nesting loca tions would be an ideal control stratagem. However, pharaoh ants primarily nest indoors, including inside wall voids, in cabinets and under sinks (Edwards 1986). These inacces sible nesting locations, coupled with the polydomous behavior of the pharaoh ant, make direct treatment of all nests nearly impossible. Management of pharaoh ant infestations includes the use of baits and residual sprays. Effective baits should possess a slow acting toxicant, be readily transferred

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2 between ants, and should not be repellent (Str inger et al. 1964). Th e bait toxicant should possess all three characteristics or it will not be effective in eliminating an infestation. Baiting can be very effective, but is also ti me consuming and can take up to 8-12 weeks to reach control (Klotz et al. 1996, Oi et al 2000). Pharaoh ants exhibit changes in food preference depending upon satiation of one food source (Edwards and Abraham 1990). This makes it difficult to control infestations with the use of a single bait formulation. The bait should also be available for a long ti me interval, and the ants must feed on it for at least 3 d (Klotz et al. 1997b). Residual insecticides provide pest control operators with anothe r treatment option. There are different treatment methods for residual insecticides depending upon the species of ant that is involved. Perimete r-invading ants, such as the Argentine ant and red imported fire ant can be excluded from a st ructure with the application of a barrier of repellent or nonrepellent inse cticides. Knight and Rust (1990) evaluated residual insecticides against la boratory Argentine ant workers and found that insectic ides high in repellency or low in repellency can still be an effective barrier treatment. Bifenthrin barrier treatments were 100% effective in excluding red imported fire ants from structures up to 15 wk after treatment (Pra nschke et al. 2003). Recently, perimeter treatments with fipronil dramatically reduced foraging activity of st ructure invading ants (Scharf et al. 2004). Since pharaoh ants are in terior nesting ants, th e use of a repellent perimeter treatment would trap the ants insi de the structure and not be an effective control measure. The use of perimeter treatme nts for control of pharaoh ants relies on the ants crossing the treatment zone. Although pharaoh ants nest indoors, Oi et al. (1994)

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3 found that in Florida, most of the foraging activity occurs outdoors. Placement of bait stations outdoors can be an effective tr eatment (Oi et al. 1994, Oi et al. 1996). If a residual treatment could mimic the charac teristics of a bait t oxicant, it could be an effective treatment strategy for pharaoh ant infestations. The resi dual toxicant should not cause foraging ants to avoid the treated su rface, it should have a delay in toxicity so the foraging ants could effectively spread th e toxicant once they reach the nest, and it should be transferable between ants via cont act or trophallaxis. Such a treatment could allow pest control operators to manage a phara oh ant infestation in a much more efficient manner. The objective of this study was to determ ine the efficacy of a residual treatment against infestations of the pha raoh ant. A repellency experiment was conducted in order to determine if foraging ants would walk on treated surfaces. An effective treatment should not repel foraging ants from cont acting a treated surf ace. A lethal time experiment was conducted on both pharaoh ant workers and queens in order to determine the time until death after exposure to the treat ment. The treatment should exhibit a delay in mortality, where death would occur in 1-5 d. Finally, a bridge experiment was conducted in order to determine effects of a residual treatment on workers and reproductives within the colony. Residual tr eatments should affect not only foraging workers but also nesting workers and reproductives located within the nest.

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4 CHAPTER 2 LITERATURE REVIEW Origin and Distribution The African-Middle Eastern region is thought to be the place of origin of this ant (Arnold 1916, Bolton 1987). Through co mmerce, it has become worldwide in distribution (Edwards 1986). Artificial heating has allowed this once tropical ant to spread to temperate regions (Harris 1991). Biology M. pharaonis belongs to the subfamily Myrmicinae, within the family Formicidae, and the tribe Solenopsidini (Bolton 2003) The workers of M. pharaonis are yellowish brown to reddish and reach approximately 2 mm in length (Vail 1996). They possess 2 nodes, and a 12-segmented antennae with a 3-se gmented club. The queens of this species are roughly twice the size of workers (4 mm) and possess a definitively larger gaster than the workers (Haack 1987). Males are black and possess wings. Colonies are polygynous. Several hundred queens can reside in one nest. M. pharaonis is closely associated with human ha bitation and is rarely found nesting outdoors (Vail 1996). Any warm area with high humidity can be utilized as a nesting location (Edwards 1986). Thus, wall voids, at tics, door housings, elec trical outlets, sinks, and other crevices within the home serve as nesting sites (Vai l 1996). Colonies are also polydomous, possessing multiple nesting locations with little or no aggression between nests (Edwards 1986, Haack 1987).

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5 Colony foundation does not occur through a singl e fertilized female, as in most ant species. Instead, reproduction occurs by so ciotomy (budding). Workers will carry brood to a separate nesting location. Queens are not necessary for the survival of the new nest, however, they will often accompany the work ers (Edwards 1986). Migrating workers may set up temporary nesting locations before se ttling at a final site. These temporary, or satellite, nests may be connected by odor tr ails and may represent a single, enormous interconnected colony (Harris 1991). Seve ral conditions can instigate sociotomy including overcrowding, envir onmental conditions, and lack of food or water (Edwards 1986). Recent evidence suggests that the application of repe llent insecticides can also provoke sociotomy (Buczkowski et al. 2005). Although, newly ecdysed, virgin queens temporarily possess wings, they are incapable of flight. Males will mate with the females from the same, or a nearby colony on the ground. This lack of outbreeding has le d to a lack of aggr ession between nests, and has helped develop the behavior of budding (Wilson 197 1). Oviposition occurs quickly after mating. In the fi rst 4-5 weeks, the queen will la y 4-6 eggs per day. The rate of oviposition then rises to 25-35 eggs per day. During the last 5 weeks of the queen’s life, the oviposition ra te drops to 3-7 eggs per day (Edwards 1986). The maximum lifespan of the queen has been measured at 39 weeks in the field (Peacock 1950) and over 52 weeks in the laboratory (E dwards 1986). The average lifes pan of a queen is about 200 days (Petersen-Braun 1975). Over her lifespan, a queen may lay over 4500 eggs (Edwards 1986). Such reproductive potential allows for the quick development of large colonies, despite high larval mortality (Peaco ck 1950). Brood is separated into different stages of development probably to cu rb larval cannibalism (Edwards 1986).

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6 Peacock recorded the following developmen t times for the worker caste: egg 7.3 days, larvae 17 days, prepupa 3.1 days, pupa 9 da ys. The average life cycle is completed at 36.4 days. The life cycle of the reproductive caste is completed at 41.25 days (1950). Subsequent studies by Petersen-Braun (1973), Kretzchmar (1973), and Berndt and Eichler (1987) have given similar results (Vail 1996). The worker caste exhibits age polyethism. The younger workers will remain in the nest and tend the brood. The older workers forage for food and water (Holldobler and Wilson 1990). Worker ants live about 9-10 weeks. The older workers constitute a small fraction (0.7 to 5.6%) of the colony due to their short lifespan (Vail 1996). Food and Foraging M. pharaonis are omnivorous and will feed on various sources of lipids, carbohydrates, and proteins (Edwards 1986). Th e larvae and queen of the colony require large amounts of protein for development and vitellogenesis (Chapman 1998). Dead insects found at lights and window sills are pr obably the most important source of this protein (Sudd 1960, Oi et al. 1994 ). Foraging workers transpor t protein back to the nest and feed it to the larvae. The larvae may help the adult workers receive nutrients that they cannot consume. Stomodeal as well as proctodeal trophallaxis from larvae to worker has been documented (Holldobler and Wilson 1990). The salivary and rectal fluid extracted from the larvae of M. pharaonis have been shown to contain nitrogen, amino acids, proteases, and carbohydrases (Holldobler and Wilson 1990). These compounds are necessary for the breakdown of worker food sources as well as the production of additional enzymes. Edwards and Abraham showed that M. pharaonis exhibits a satiation and altern ation response to different food sources. When presented with a highly attractive food for several week s, foraging ants will consume an alternative

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7 food source when given a choice (1990). This behavior allows the colony to receive a balanced diet. Worker ants display various foraging behavi ors. Initial foraging consists of worker ants wandering in search of food. Foraging ants will often orient along a structural guideline (Klotz and Reid 1992). Upon finding f ood, the worker will return to the nest in the straightest possible path (Klotz 2004). While returning, the ant will lay a trail pheromone, which will lead additional workers straight to the food source. The trail pheromone has been identified as fa ranal, (6E, 10Z)-3,4,7,11-tetramethyl-6,10tetradecadienal (Ritter et al 1977, Holldobler and Wilson 1990). Beekman et al. (2001) showed that the use of trail pheromones can only be sustained in la rge colonies (over 600 ants). Smaller colonies cannot sustain or dered foraging behavior, possibly due to the ephemeral nature of the trail pheromone. J eanson et al. (2003) s howed that the trail pheromone decays in 10 minutes on a plastic su rface. If additional ants do not reinforce the trail, it will disappear. Smaller colonies will typically forage in a random wandering fashion. Medical Importance For many years, the importance of M. pharaonis has been centralized on its pest status. Potential for disease transmission has increased the importance of this ant. In 1972, worker ants were shown to transport Pseudomonas, Staphylococcus, Salmonella, Clostridium, and Streptococcus (Beatson). Additionally, the pharaoh ant has been shown to vector Bordetella bronchiseptica, a swine pneumonia (Beatson 1972). Hospitals, including intensive care units have been in fested. Recently, Burrus (2004) showed that pharaoh ants will readily consume many fluids found in hospital environments, including dextrose, Ensure, plasma, and saline.

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8 Control Pharaoh ants are among the most difficult pe sts to manage effec tively. Their social status makes them especially cumbersome fo r both homeowners as well as pest control operators. Several factors acc ount for this including their polydomous nesting behavior and polygyny. The most effective method of elim inating an ant infestation is to treat the nest directly. However, this becomes extremely difficult when the ants have multiple nesting locations which are linked and shar e resources (Vail 1996) Since pharaoh ant colonies typically have several hundred qu eens, management efforts must include methods of delivering insectic ide to the reproductives. Management of pharaoh ant infestations must include identification, monitoring, and implementation of an integrated pest ma nagement program. Proper identification is essential. Pharaoh ants are very similar in appearance to fire ants as well as other perimeter-invading ants. If pharaoh ants were misidentified as a perimeter-invading ant, effectively eliminating an infestation would be difficult. Proper monitoring can help to ensure that treatment methods are worki ng properly. Integrated pest management methods include exclusion, physical removal, sanitation, and chemical management. Exclusion is important for keeping ants from entering a structure. Cracks and crevices should be properly sealed. Window screens should be free of tears. Although this will not remove an existing infestati on, it may help prevent ants from colonizing inside. Sanitation is crucial for proper management of ant infestations. Ants are extremely susceptible to dehydration. Leaky faucets must be fixed and extraneous sources of water must be eliminated. Crumbs and food spill s should be cleaned immediately. Without proper sanitation, ant infestations cannot efficiently be managed.

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9 Chemical management methods for pharaoh ant control have em ployed the use of residual sprays and toxic baits. In the past, repellent residual sprays have been used. However, such compounds will only k ill the foraging workers (Williams 1990, Vail 1996). This represents only 0.7 to 5.6% of the entire colony (V ail 1996). Repellent sprays may also lead to the formation of satellite nests (Va il and Williams 1994, Vail 2002, Buczkowski et al. 2005). Thus, repe llent compounds may actually promote dispersion and growth of a pharaoh ant infestation. Colonies of M. pharaonis are able to resist starvation for prolonged periods of time with the aid of larval trophallaxis (Holldobler and Wilson 1990). Thus, even if a repellent spray were to cut a colony off from its food source, it could still survive for long periods of time. It is generally believed that baits are the only control method that will achieve complete eradication of phara oh ant infestations (Klotz et al. 1996, Klotz 2004). Baiting takes advantage of the fact that ants will fora ge and recruit to food sources. Bait is placed in cracks, crevices, along ac tive trails, and other possible ant congregation areas. The foraging ants feed on the bait and transmit th e toxicant to nest mates via trophallaxis. Baits employ the use of insect growth re gulators or stomach poisons (Vail 1996). Formulations for baits include granular, ge l, liquid, and stations Hooper-Bui et al. (2003) found that pharaoh ants prefer granul e sizes between 420-590 micrometers. Most granular bait particles are 1,000 to 2,000 microm eters, however, Niban particles are less than 420 micrometers (Hooper-Bui et al. 2003). Current baits include the following: Bayer Maxforce Ant Killer Gr anular bait (Hydramethylnon), Ni ban Puffer Fine Granular Bait (Orthoboric acid), Whitmire Micro-ge n Advance Ant Bait (Abamectin). When

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10 applying a granular bait, it wa s found that dispersion pattern is not important (Silverman and Roulston 2003). Gel baits are a very popular formulation w ith pest control operators, however, it was found that consumption of liquid bait is hi gher than gel formulated bait in Argentine ants (Silverman and Roulston 2001). A lthough gel baits are more convenient for transport, application, and longe vity, they may not be as eff ective as a liquid formulation (Silverman and Roulston 2001). Currrent gel baits include: Gourmet Ant Bait (Disodium octaborate tetrahydrate), Bayer Ma xforce FC Ant Killer Bait Gel (Fipronil), Intice Sweet Ant Gel (Orthoboric acid), a nd Drax Ant Bait (boric acid). Liquid boric acid baits have been evaluate d against colonies of ants many times (Klotz et al. 1996, Klotz et al 1997a, b, Klotz et al. 1998, Klot z et al. 2000, Rust et al. 2004). Liquid baits are gene rally found to reduce the work er population and foraging activity after 2-8 wks. Commercial liqui d baits include Whitmire Micro-Gen Advance Liquid Ant Bait (boric acid), Terro Ant Killer II (Disodium octaborate tetrahydrate), and Uncle Albert’s Liquid ant bait (boric acid). Baits are also formulated as bait stations Bait stations often employ liquid or gel bait within a plastic container. This method keeps the toxicant out of reach of children and pets. Current bait stations incl ude: FMC Fluorguard Ant Bait Stations (Sulfluramid), Bayer Maxforce Ant Bait Stations (Hydramethylnon), Whitmire Microgen DualChoice Ant Bait Stations (Sulfl uramid), and Raid Max Ant Bait. Oi et al. (2000) showed th at insect growth regulator baits, although slower acting, allow for greater transfer of the toxi cant among the colony. Currently, Pharorid

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11 (methoprene) is an insect grow th regulator labeled for pharaoh ant infestations. Pharorid is not formulated as a pre-mixed bait, and can be combined with any type of food source. Although pharaoh ants nest indoors, it was found that in Florida, most of the foraging activity occurs outdoors (Oi et al. 1994). Placement of bait stations outdoors can be an extremely effective treatment (Oi et al. 1994, Oi et al. 1996, Vail et al. 1996). If infestations could be elimin ated with an outdoor treatmen t, both pest control operators as well as homeowners would benefit. The pe st control operator coul d evaluate and treat the structure without intruding or pl acing chemicals within the home. The use of perimeter treatments for contro l of ant infestations has been evaluated many times (Knight and Rust 1990, Oi et al. 199 4, Oi et al. 1996, Rust et al. 1996, Vail et al. 1996, Pranschke et al. 2003, Scharf et al. 2004, Soeprono and Rust 2004a, Soeprono and Rust 2004b, Buczkowski et al. 2005). Perime ter treatments with residual insecticides have become an important tool for pest contro l operators. Bifenthrin barrier treatments have been shown to be 100% effective in exclud ing red imported fire ants from structures up to 15 wk after treatment (Pranschke et al 2003). Knight and Rust (1990) evaluated several residual insecticides against laborat ory Argentine ant workers for repellency and efficacy. Their results indicated that efficacy was not necessarily dependant upon repellency. When workers encountered less repellent compounds, they were more likely to receive a higher dose of the insecticide. Nonrepellent insecticides have increased the popularity of peri meter treatments. Such compounds rely on the ants’ inability to detect the presence of the chemical. The ants walk through the treatment zone, and even tually gather enough toxi cant and die. In the process, the toxicant is transferred to nestmates thr ough social grooming, trophallaxis,

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12 necrophoresis, and other nestmate interactions. Such horizontal tran sfer of insecticide greatly increases the effectiv eness of a perimeter treatment (Soeprono and Rust 2004a). Recently, perimeter treatments w ith fipronil have been show n to dramatically affect foraging activity of structure invading ants (S charf et al. 2004). Mu ch like bait toxicants, fipronil can be horizontally transferred be tween nest mates, thus increasing its effectiveness (Soeprono and Rust 2004a). The efficacy of a residual treatment for management of an ant infestation is dependant upon many factors, including, the t ype of surface the treatment is applied to, nesting location of the ants, formulation of the insecticide, transferability of the compound, and delay in toxicity of the compound (Chadwick 1985, Knight and Rust 1990, Osbrink and Lax 2002, Soeprono and Ru st 2004a, Soeprono and Rust 2004b).

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13 CHAPTER 3 MATERIALS AND METHODS Insects Pharaoh ant colonies were rear ed at the University of Fl orida (Gainesville, FL) in a rearing room maintained at 27 1.5C, 40 7% RH. Colonies were provided ad libitum with water, 10% sugar water, and fre shly killed American cockroaches ( Periplaneta americana), obtained from the Urban Entomology laboratory, University of Florida, Gainesville, FL). Plastic trays (56 by 44 by 12 cm; Panel Controls, Greenville, SC) coated on the inne r walls with Fluon (AGC Chemicals, Bayonne, NJ) served as rearing containers. Nest cells consisted of square plaster-filled (Dentsply, York, PA) plastic Petri dishes (100 x 15mm; Becton Dickinson Labw are, Franklin Lakes, NJ)(Williams 1990). A soldering iron was used to melt a 3 mm hole in the center of each side of the bottom dish to allow the ants access. Yellow acetate paper covered the nest cell to filter light and make it more attractive for brood rearing. Insecticide Treatments. Treatments consisted of fipronil (Termidor SC 9.1% [AI]; BASF Research Triangle Park, NC), lambda-cyhalothrin (Demand CS 9.7% [AI]; Syngenta, Wilmington, DE), and deionized water as a control. Insecticides were mixed resulti ng in solutions equivalent to the manufacturer’s label rates for ant contro l (0.06% for fipronil and 0.015% for lambdacyhalothrin). Testing areas were placed within a 929 cm2 paper sheet and 3.78 ml of solution was applied with an airbrush (Paas che, Type H, Chicago, IL) to the entire surface of the sheet. All te sting areas were allowed to dry for 24 h prior to use.

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14 Repellency Assay Arenas consisted of three Fluon rimmed polystyrene Petr i dishes (100 by 15 mm; Fisher Scientific, Pittsburgh, PA) connected w ith a wire mesh bridge (0.64 cm mesh, 23 gauge; LG Sourcing, North Wilkesboro, NC). The wire mesh was cut into a ‘Y’ shape (15 by 10 cm) and bent to allow the ants access from the untreated release dish to any of the arenas. One of the arenas was treated with deionized water while the other was treated with either fipronil or lambda-cyhalo thrin. Mortality and location of live ants were recorded in each arena every 15 min for 1 h. Repellency was defined as the mean percentage of live ants in the untreated aren a (Appel et al. 2004). Six replicates were performed for each treatment with fifty ants in each replicate. Lethal Time Assay Treatments were applied to glass (297 by 50 by 3 mm) or painted plywood panels (297 by 50 by 10 mm; White Flat Latex Exteri or; Masco, Santa Ana, CA). Four Fluon coated CPVC pipe pieces (40 by 40 cm) were hot -glued to the midline of each panel at 5 cm increments. Ten worker and ten queen ants were placed in each pipe piece. Mortality and knockdown were recorded every 15 min for 2 h and every 30 min thereafter for 12 h or until all ants were dead for up to 5 d. In cases where ants were still alive at 12 h, observations were suspended and resumed 24 h after treatment. Recording of control mortality ceased when the last ants in th e treatments died. Knockdown was defined as the inability of the an t to right itself, and mortality was defined as having no response when probed. Six replications per panel type per treatment type were conducted for a total of 36 experimental units.

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15 Bridge Assay Experimental colonies consisted of 1000 worker ants (500 selected from outside nest cells and 500 selected fr om inside nest cells), 12 queens, and 200 mg of brood held in Fluon coated plastic trays (56 by 44 by 12 cm; Panel Controls, Greenville, SC). The 500 outside worker ants were placed directly in the tra y, while queens, brood, and 500 inside ants were placed into plaster-fille d polystyrene Petri dishes (60 by 15 mm; Becton Dickinson Labware, Franklin Lakes, NJ) whic h served as nest cells. Nest cells were covered with yellow acetate paper and placed at one end of the tray. Water and 10% sugar water were provided ad libitum Experimental colonies were allowed to acclimate for 24 h. After the 24 h acclimation period, a deli cup, (237 ml; Fabri-Kal, Kalamazoo, MI) which had been coated on both vertical sides with Fluon, was centered at the opposite end of the tray from the nest cell, 7 cm from the front. A wood support (6 by 5 cm) was placed inside the Fluon coated cup. A second wood support was placed near the nest cell, 197 mm from th e first support (see Fig. 2-2). Treated glass (297 by 50 by 3 mm) or treated painted plywood bridges ( 297 by 50 by 10 mm) were placed on the two wood supports. The Fluon coated cup prevented ants fr om entering or exiting the cup without crossing the bridge (see Fig. 2-3). To provide food and induce foraging, a dead adult cockroach was placed inside the deli cup every other day th roughout the duration of the experiment. Worker and queen mortalit y was recorded at 1 h, and at 1, 3, 7, 14, and 21 d. Moribund (unable to right themselves) ants were considered dead. Dead ants were removed at the time of counting. Digital photog raphs were taken of the nest cell at each observation period prior to removal of dead ants for the wood bridge assay (Sony Cybershot model number DSC-W1; Sony, Pittsbur g, PA). Additionally, brood was weighed

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16 prior to treatment and 28 d after placement of bridges. Each treatment was replicated six times per surface type. Data Analysis Numbers of live and dead ants in each of the three Petri dishes were analyzed by one-way analysis of variance, with means se parated by Student Newman-Keuls test when P values of the analysis of variance were signi ficant. Percentage of responding ants that were located in the treated di sh was calculated for each treat ment. A paired one-tailed ttest ( = 0.05, SAS Institute 2002) was used to determine whether the percentage of responding ants differed significantly from 50% (Ho: Percentage 50% = 0). For the lethal time assay KD50 and LT50 values were estimated by pr obit analysis of correlated data: multiple observations at one concentration (Throne et al. 1995), SAS Institute 2002). Significant differences were determ ined by nonoverlap of the 95% confidence intervals (CI). For the bridge assay, cumu lative worker and queen mortalities for each treatment were analyzed with a one-way analys is of variance at each time after treatment, and means were separated using Student Newman-Keuls test when P values for the analysis of varian ce were significant ( = 0.05, SAS Institute 2002).

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17 Figure 3-1. Repellency assay se tup showing location of rele ase, treated, and untreated dishes. A ‘Y’ shaped bridge of steel mesh was used to connect all three dishes. The inner rims of all dishes were coated with Fluon to prevent ant escape. 50 ants were placed into releas e dish and allowed to move to any of the three dishes. Untreated dish (100 mm diameter; 15 mm deep) Treated dish (100 mm diameter; 15 mm deep) Release dish (100 mm diameter; 15 mm deep)

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18 Figure 3-2. Bridge assay setup showing location of nest cell, bridge, and food sources. A dead adult cockroach was placed into the Fluon coated cup every other day. The nest cell held 12 queens, 200 mg brood, and 500 nesting workers. 500 foraging workers were placed in the center of the arena. Sugar water vials (15 ml) Water vials (15 ml) Nest cell (60 mm diameter; 15 mm deep) Fluon coated cup (237 ml) 56 cm Treated bridge 44 c m 297 mm 50 mm

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19 Figure 3-3. Glass bridge assay setup showing ants trailing along bridge.

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20 CHAPTER 4 RESULTS Repellency Assay In all treatments, many of the ants immedi ately left the release dish and began to explore the other dishes. An ts were observed walking on all dishes in all treatment groups. There was no significant difference am ong the distribution of live ants in the untreated and treated dishes for either the fipronil or the lambda -cyhalothrin treatments throughout the duration of the repellency assay (Table 4-1). There were a large number of dead ants in the lambda -cyhalothrin treated dish at 30, 45, and 60 min (Table 4-1). Results from the t-test indicated that si gnificantly fewer than 50% of responding ants were in the fipronil treated di sh at 15 min (t value = 2.69), indicating sl ight initial repellency. Fipronil was not repellent at 30, 45, or 60 min. Results from the t-test indicate that lambda-cyhalothrin was not repe llent to worker pharaoh ants at any time after treatment (Table 4-2). Lethal Time Assay In all treatments, worker ants clustered together in small groups after placement on the treatment. On the glass surface work ers began to exhibit grooming behavior, including antennal and leg cl eansing at ~10 min in the lambda-cyhalothrin group and after ~30 min in the fipronil group. When pharaoh ant workers were exposed to glass and wood treated with lambda-cyhalothrin, KD50 values for both glass and wood surfaces were <1 h. Fipronil treatmen t caused significantly higher KD50 values than lambdacyhalothrin on glass and wood surfaces. Fipron il applied to wood was the only treatment

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21 that caused delayed knockdown. W ood treated with fipronil had a KD50 value of >24 h (1,950.00 min). LT50 values for pharaoh ant workers on lambda -cyhalothrin ranged from 2 to 3 h on glass and wood surfaces respectively (Table 4-4) When ants were exposed to fipronil, the LT50 value was 496.54 min (8.3 h) on glass, and almost five times that on wood (39.9 h). A delay in mortality for >24 h only o ccurred in the fipronil treatment on the wood surface. There was no worker mortality on water treated surfaces in the lethal time assay. LT50 values for pharaoh ant queens were only sl ightly higher than those of workers when exposed to lambda-cyhalothrin on both glass and wood (Table 4-4). There was no significant difference in LT50 values between pharaoh ant workers and queens exposed to lambda-cyhalothrin on wood. LT50 values for pharaoh ant queens exposed to fipronil on glass were only slightly higher than LT50 values for workers (521.93 min for queens and 496.54 min for workers). On the wood surface, however, LT50 values for queens were much higher than for workers (4,422.00 min for queens vs. 2,396.00 min for workers). There was no queen mortality in the control lethal time assay. Bridge Assay After placement of the cockroach as food, an ts were seen foraging across the bridge within minutes. Within 30 min, a distinct trail of ants could be seen in most replicates. In the glass bridge assay, ants were quickl y knocked down and began to die within 1 h in the treatment groups. Over 50% of the worker population was dead at 1 d for both of the treatments (62.13% for fipronil and 54.90% fo r lambda-cyhalothrin). The control setup never exceeded 50% mortality for the worker population for the duration of the test. Nearly 80% of the worker population was dead at 14 d in the fipronil treated glass setup. Similarly, the lambda-cyhalothrin treated gla ss setup resulted in 75% worker mortality

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22 after 21 d (Table 4-5). Mortality for the wate r treated glass bridge was significantly less than both the fipronil and la mbda-cyhalothrin groups at time 1 h and remained so throughout the duration of the experiment. Worker mortality on the wood surface exhibi ted a delay in comparison to the glass surface. Mortality was significantly higher in the lambda-cyhalothrin treatment (28.83 %) than either the c ontrol (13.37 %) or fipronil (15.88 %) treatment at 1 d. Mortality after 1 d increased only slightly in the lambda-cyhalothrin treatment. The fipronil treatment resulted in significantly higher mortality (40.38 %) than lambda-cyhalothrin (31.20%) and the control (20.25 %) at 3 d and re mained higher thereafter. At 14 and 21 d, the lambda-cyhalothrin group was not signifi cantly different than the control group (Table 4-5). The fipronil treatment re sulted in 100% mort ality after 21 d. Queens generally remained inside the nest cells for the duration of the experiment. However, in the glass bridge setups, a few qu eens were seen foraging at the 14 d mark in two replicates of the fipronil treatment. In the glass brid ge setup, no significant queen mortality was observed throughout the duration of the experime nt (Table 4-6). In the wood setup, however, the fipronil treatmen t exhibited significantly greater queen mortality at 14 d (59.73%) and 21 d (77.78%). The mass of brood (by weight) decreased for fipronil and lambda-cyhalothrin treatments. Fipronil treated glass resulted in a 43.55% re duction in brood mass (Table 47). Lambda-cyhalothrin resulted in a 52.28% reduction in brood mass. The mass of brood in the fipronil and lambda-cyhalothrin trea tments were not significantly different at 28 d after placement of the treated bridges. In the wood bridge experiment, fipronil exhibited a 79.20% reduction in mass of br ood at the end of the experiment. The

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23 Lambda-cyhalothrin also exhi bited a significant difference in mass of brood compared to the control 28 d after placement of the trea ted bridges (Table 4-7). However, this reduction was small in comparison to the redu ction in brood in the fipronil treatments (17.19% for lambda-cyhalothrin). The photographs of two of the nest cells from each treatment for the wood bridge assay can be seen in Figs. 4-1 through 4-6. These present a representation of each of the treatment groups. No visible difference was obs erved in the control replicates throughout the duration of the experiment. A visible reduction of activity was observed in the lambda-cyhalothrin replicate 4 (see Fig. 4-4). However, queens and brood are still visible 21 d after placement of the treated bridge. In the fipronil replicates, there is a strong reduction in worker and queen number, as we ll as a reduction in brood. After 21 d, there are very few queens and brood re maining (Figs. 4-5 and 4-6).

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24Table 4-1. Distribution of 50 work er ants in release, untreate d, and water, fipronil, or lam bda-cyhalothrin treated Petri dish es Number of ants (mean SE) at time (min) 15 30 Treatment Dish Live Dead Live Dead Control Water-treated 10.00 1.53a 0.00 0.00 9.33 0.67a 0.00 0.00a Untreated 8.17 1.42a 0.00 0.00 10.67 0.56a 0.00 0.00a Release 31.83 1.33b 0.00 0.00 30.00 1.15b 0.00 0.00a Fipronil Treated 6.33 1.26a 0.00 0.00 9.83 1.62a 0.00 0.00a Untreated 10.00 1.06a 0.00 0.00 11.17 2.06a 0.00 0.00a Release 33.67 1.82b 0.00 0.00 29.00 1.71b 0.00 0.00a -cyhalothrin Treated 5.67 1.28a 0.00 0.00 6.50 1.34a 13.00 3.46b Untreated 6.00 1.59a 0.00 0.00 6.33 1.65a 0.00 0.00a Release 38.33 1.23b 0.00 0.00 24.17 1.87b 0.00 0.00a Number of ants (mean SE) at time (min) 45 60 Control Water-treated 9.50 0.76a 0.00 0.00a 9.50 0.76a 0.00 0.00a Untreated 9.17 0.54a 0.00 0.00a 8.83 0.60a 0.00 0.00a Release 31.33 0.88b 0.00 0.00a 31.67 0.95b 0.00 0.00a Fipronil Treated 9.33 1.52a 0.00 0.00a 11.00 0.97a 0.00 0.00a Untreated 9.67 1.33a 0.00 0.00a 10.33 0.99a 0.00 0.00a Release 31.00 1.69b 0.00 0.00a 28.67 1.41b 0.00 0.00a -cyhalothrin Treated 4.33 0.56a 18.50 6.02b 3.50 0.50a 23.67 4.03b Untreated 4.67 0.76a 0.00 0.00a 2.17 0.48a 0.00 0.00a Release 22.50 1.28b 0.00 0.00a 20.67 1.74b 0.00 0.00a Means followed by the same letter within the same colu mn treatment group are not significantly different (p=0.05, Student Newman-Keuls test, SAS Institute, 2002).

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25 Table 4-2. Repellency of fipron il or lambda-cyhalothrin treated glass to pharaoh ant workers _______________________________________________________________________ Time (min) Treatment n % Responding T statistic P in Treated dish _______________________________________________________________________ 15 Fipronil 6 38.76 2.69 0.04 -cyhalothrin 6 48.59 -0.05 0.96 30 Fipronil 6 46.81 0.39 0.71 -cyhalothrin 6 50.66 -0.21 0.83 45 Fipronil 6 49.11 0.16 0.88 -cyhalothrin 6 48.11 0.50 0.64 60 Fipronil 6 51.57 -0.49 0.64 -cyhalothrin 6 61.73 -1.52 0.19 _______________________________________________________________________ Ha: Proportion of ants in treated dish 50% 0; One tailed t-test (SAS Institute, 2002)

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26Table 4-3. KD values of pharaoh ant workers when exposed to glass or wood treated with fipronil or lambda-cyhalothrin ____________________________________________________________________________________________________________ Treatment Surface na Slope KD50 min (95% CI) KD90 min (95% CI) 2 P ____________________________________________________________________________________________________________ Fipronil Glass 1,680 3.08 0.20 194.98 (182.15-211.58) 508.71 (432.51-625.77) 6.37 0.27 Wood 1,680 4.64 0.25 1,950.00 (1,887.00-2,019.00) 3,685.00 (3,408.00-4,055.00) 4.56 0.47 -cyhalothrin Glass 720 4.82 0.30 19.07 (18.11-20.17) 35.20 (32.11-39.43) 0.186 0.67 Wood 1,440 1.96 0.14 46.72 (42.23-52.79) 210.34 (161.81-297.11) 3.44 0.49 ____________________________________________________________________________________________________________ aTotal number of trials with 240 ants per trial (Probit [SAS Institute 2002]).

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27Table 4-4. LT values of pharaoh ant workers and queens when expos ed to glass or wood treated with fipronil or lambda-cyhalothr in ____________________________________________________________________________________________________________ Caste Treatment Surface na Slope SE LT50 min (95% CI) LT90 min (95% CI) 2 P ____________________________________________________________________________________________________________ Worker Fipronil Glass 1,200 18.29 1.14 496.54 (491.47-501.40) 583.49 (572.92-596.88) 5.14 0.16 Wood 960 7.98 0.40 2,396.00 (2,324.00-2,471.00) 3,468.00 (3,321.00-3,648.00) 4.57 0.12 -cyhalothrin Glass 1,440 4.00 0.21 137.84 (131.67-144.92) 288.12 (261.42-324.18) 3.85 0.42 Wood 960 3.30 0.27 158.24 (149.25-167.94) 386.82 (336.85-466.66) 0.17 0.92 ____________________________________________________________________________________________________________ Queen Fipronil Glass 960 12.52 0.64 521.93 (512.98-531.05) 660.68 (642.96-682.15) 3.58 0.17 Wood 720 14.53 1.41 4,422.00 (4,343.00-4,520.00) 5,418.00 (5,185.00-5,770.00) 0.13 0.72 -cyhalothrin Glass 1,440 4.18 0.42 164.45 (148.15-191.26) 333.38 (268.61-459.48) 7.05 0.42 Wood 1,440 5.98 0.28 172.13 (167.12-177.16) 282.05 (269.33-297.55) 6.52 0.16 ____________________________________________________________________________________________________________ aTotal number of trials with 240 ants per trial (Probit, SAS Institute 2002).

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28 Table 4-5. Percent mortality of pharaoh ant work ers in response to placement of glass or wood bridges treated with water, fipronil, or lambda-cyhalothrin ____________________________________________________________________________ Percent Mortality SE ______________________________________________ Surface Time na Control Fipronil -cyhalothrin ____________________________________________________________________________ Glass 1 h 6 0.00 0.00a 40.18 5.29b 36.92 5.65b 1 d 6 8.78 0.871a 62.13 5.07b 54.90 4.60b 3 6 14.60 0.81a 68.70 0.55b 59.38 0.62b 7 6 21.27 0.62a 72.70 0.54b 62.12 0.19b 14 6 33.87 2.58a 79.90 1.62b 68.42 0.81b 21 6 43.07 1.35a 86.90 1.10b 75.73 0.33b Wood 1 h 6 0.00 0.00a 11.32 0.30b 10.03 0.23b 1 d 6 13.37 0.63a 15.88 0.96a 28.83 0.56b 3 6 20.25 1.07a 40.38 1.95b 31.20 0.72c 7 6 38.65 1.30a 60.48 0.90b 45.50 0.69b 14 6 52.87 1.24a 84.25 3.73b 56.98 1.73a 21 6 57.30 0.03a 100.00 3.50b 63.13 1.15a _________________________________________________________________________________________________________ a Number of replicates with 1000 worker ants per replicate Treatments applied at label rates for ant c ontrol; fipronil 0.06%, and lambda-cyhalothrin 0.015% Means within a row followed by the same letter are not significantly different (P=0.05, Student Newman-Keuls test, SAS Institute 2002).

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29 Table 4-6. Percent mortality of pharaoh ant queens in response to placement of glass or wood bridges treated with water, fipronil, or lambda-cyhalothrin ___________________________________________________________________ Percent Mortality SE _________________________________________ Surface Time na Control Fipronil -cyhalothrin _______________________________________________________________________ Glass 1 h 6 0.00 0.00 2.83 2.83 0.00 0.00 1 d 6 0.00 0.00 2.83 2.83 0.00 0.00 3 6 4.00 1.79 5.50 5.50 1.33 1.33 7 6 5.33 1.69 7.00 7.00 1.33 1.33 14 6 6.83 2.59 13.83 12.30 1.33 1.33 21 6 6.83 2.59 16.50 13.40 1.33 1.33 Wood 1 h 6 0.00 0.00 0.00 0.00 0.00 0.00 1 d 6 0.00 0.00 0.00 0.00 0.00 0.00 3 6 0.00 0.00 1.38 1.38 0.00 0.00 7 6 0.00 0.00 6.93 3.98 0.00 0.00 14 6 1.38 1.38a 47.20 11.73b 2.77 1.75a 21 6 1.38 1.38a 77.73 10.02b 11.08 4.65a ____________________________________________________________________ a Number of replicates wi th 12 queens per replicate Treatments applied at label rates for ant c ontrol; fipronil 0.06%, and lambda-cyhalothrin 0.015% Means within a row followed by the same letter are not significantly different (P=0.05, Student Newman-Keuls test, SAS Institute 2002).

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30Table 4-7. Mass of brood before and after placement of bridges treated with fipronil or lambda-cyhalothrin Brood mass (mg SE) Surface Time (d) n Control Fipronil -cyhalothrin _____________________________________________________________________________________ Glass 0 6 200.33 0.16 200.00 0.21 200.10 0.15 28 6 194.68 12.80a 112.90 10.82b 95.48 12.54b Wood 0 6 200.52 0.18 200.12 0.32 200.30 0.19 28 6 214.90 16.93a 41.63 11.49b 165.57 17.31c ______________________________________________________________________________________ Means within a row followed by the same letter are not significantly different (P=0.05, Student Newman-Keuls test, (Institute 2002).

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31 A B C D Fig. 4-1. Series of photographs of Control (replicate 1) showi ng nest cell over time. A) 1 d before placement of bridge, B) 7 d af ter placement of bridge, C) 14 d after placement of bridge, D) 21 d after placemen t of bridge. Notice there is not a visible difference in activ ity from photograph A.

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32 A B C D Fig. 4-2. Series of photographs of Control (replicate 2) showi ng nest cell over time. A) 1 d before placement of bridge, B) 7 d af ter placement of bridge, C) 14 d after placement of bridge, D) 21 d after placemen t of bridge. Notice there is not a visible difference in activity, brood, or number of queens from photograph A.

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33 A B C D Fig 4-3. Series of photographs of lambda-cyhalo thrin (replicate 1) showing the nest cell over time. A) 1 d before placement of tr eated bridge, B) 7 d after placement of treated bridge, C) 14 d after placement of treated bridge. Notice a slight reduction in activity and brood, D) 21 d afte r placement of treated bridge. Notice there is a slight increase in activity fr om photograph C, and there is not a large difference in activity, brood, or queen number from photograph A.

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34 A B C D Fig 4-4. Series of photographs of lambda-cyha lothrin (replicate 2) showing nest cell over time. A) 1 d before placement of treated bridge, B) 7 d after placement of treated bridge, C) 14 d after placement of treat ed bridge. Notice a reduction in activity and brood, D) 21 d after placement of tr eated bridge. Notice an increase in activity and brood from that of photograph C. Also notice there is no queen mortality.

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35 A B C D Fig. 4-5. Series of photographs of fipronil (rep licate 4) showing nest cell over time. A) 1 d before placement of treated bridge, B) 7 d after placement of treated bridge. Notice a reduction in activity and several dead queens, C) 14 d after placement of treated bridge. Notice a reduction in ac tivity, brood, and dead queens, D) 21 d after placement of treate d bridge. Notice a reducti on in activity, brood, and only 2 remaining queens.

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36 A B C D Fig. 4-6. Series of photographs of fipronil (rep licate 5) showing nest cell over time. A) 1 d before placement of treated bridge, B) 7 d after placement of treated bridge. Notice a reduction in activity and a few d ead queens, C) 14 d after placement of treated bridge. Notice several dead queen s, D) 21 d after placement of treated bridge. Notice the reduction in brood and lack of queens.

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37 CHAPTER 5 DISCUSSION Foreword Pharaoh ants are social ins ects that can be extremely di fficult to control. They exhibit many behaviors common to social insects including divi sion of labor, trail following and recruitment, and communication. These characteristics make control very difficult. A small proportion of workers se rve as foragers (Williams 1990, Vail 1996). Treatments that aim to merely kill foraging workers will not be effective in eliminating colonies. Additionally, pharaoh ants nest i ndoors, further hindering control efforts. Treatments useful in excluding perimeter-i nvading ants may only cause trapping and budding of pharaoh ant colonies inside (V ail 1996, Buczkowski et al. 2005). Tests were performed to determine repellency, speed of kill, and the effects of a residual treatment against colonies of the phara oh ant. For a residual treatment to be an effective control measure for pharaoh ants, the insecticide should mimic the characteristics of effective bait s. Effective baits should not cause foraging ants to avoid the treatment, they should exhibit a delay in mortality of >24 h, and they should be transferable from foraging worker s to the rest of the colony. For a residual treatment to be effective, the foraging worker ant must be willing to walk onto a surface treated with the insecticide. An eff ective residual treatment for control of pharaoh ant infestations should not only be nonrepellent but the insecticide should also exhibit a delay in mortality. When toxicants are encountered in smaller concentrations, foraging ants have a longer peri od of time to transverse the treatment and

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38 return to the nest. Delay in knockdown is al so critical for spread of toxicant throughout the population. If the ants are moribund, they will not be as effective in transferring toxicant. Repellency Assay In this study, both fipronil and lambda-cyha lothrin treatments were not avoided by worker ant movements. The lack of repell ency of fipronil is in agreeance with many studies on the nonrepelle nt action of fipronil to insect s (Osbrink and Lax 2002, Ibrahim et al. 2003, Scharf et al. 2004, Soeprono a nd Rust 2004b, Buczkowski et al. 2005, Hu 2005). The lack of repellency of lambda-cyhalo thrin to pharaoh ant wo rkers is consistent with observations in other studies concerning the lack of repellency of ants to certain pyrethroids. Pranschke et al (2003) observed that red im ported fire ants were not repelled by bifenthrin. Cons equently, treatments of fi pronil and lambda-cyhalothrin would not be avoided by worker ants and w ould cause worker ant mortality when applied to surfaces in and around homes and structures. It can be difficult to determine repellency of insecticides to ants. Many behavioral aspects of pharaoh ants as well as characteri stics of the insecticides can have drastic influences on the interpretation of repellency. Such factors include the speed of kill of the insecticides, nest relocation by the ants, formulation of the insecticides, and cessation of trailing and recruitment. The speed of kill of insecticides can affect the interpretation of repellency of the chemical. Fast-acting insecticides, such as pyrethroids, cause the insects to die very quickly and confound the data. Many of the an ts will die on the treated surface, giving the notion that there is a preference for the tr eated surface (Table 4-1). Our analysis was modified to account for mortality when consid ering repellency. The distribution of live

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39 ants was equal for treated and untreated dishes so the treatments were interpreted as being nonrepellent. Pharaoh ants can be easily induced to relo cate their nests. Bu czkowski et al. (2005) found that pharaoh ants generally failed to relocate their nests under tile treated with cypermethrin or bifenthrin. This was attri buted to moderate repellency. However, more information is necessary to determine if this is due to preferentia l nesting locations. Buczkowski et al. (2005) also observed that ph araoh ants relocated their nesting location 35 d after exposure to cypermethrin. This may not necessarily be due to repellency of the compound, however, since many factors can lead to nest abandonme nt (Holldobler and Wilson 1990, Harris 1991, Buczkowski et al. 2005) My studies did not evaluate fipronil or lambda-cyhalothrin for nest ab andonment or nest relocation. The formulation of an ins ecticide can have a strong in fluence on its degree of repellency to ants (Knight and Rust 1990). Wettable powders were ranked as the most repellent formulation to Argentine ants, followed by emulsifiable concentrates, and granules (Knight and Rust 1990). In my studies, the lambda-cyhalothrin (Demand CS) used was a microencapsulated formulation. The microencapsulation is thought to protect the pyrethroid from environmental degrada tion. It may also serve to render the pyrethroid nonrepellent. However, more re search needs to be conducted on different formulations of lambda-cyhalothrin to determine if this is true. The cessation of trailing and recruitment behavior can give a false sense of repellency. Soeprono and Rust (2004) suggest ed that pyrethroids do not repel Argentine ants, but merely inhibit their ability to form recruitment trails. My results from the repellency assay demonstrate that lambda-c yhalothrin exhibits no repellency to foraging

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40 pharaoh ant workers for up to 60 min (t value = 1.52 at 60 min). Pharaoh ants were seen immediately vacating the release dish and exploring both the treat ed as well as the untreated dish. No food was provided in either dish and thus recruitment trails were not formed. Lethal Time Assay Insecticides can have varying degrees of speed of kill. Fast-acting insecticides can be effective in killing foraging ants and ex cluding ants from stru ctures. Slow-acting insecticides are more effective in elimin ating colonies. Pharaoh ant workers were knocked down very quickly when exposed to glass or wood treated with lambdacyhalothrin (KD50 values = 19.07 and 46.72 min, respectively). Although the ants were quickly knocked down, it took several hours for d eath to occur. This was evident in both the glass and wood treated la mbda-cyhalothrin assays (LT50 values = 137.84 and 158.24 min, respectively). These results agree with previous studies w ith Argentine and red imported fire ants, where pyrethroids act ve ry quickly (Pranschke et al. 2003, Soeprono and Rust 2004b). Smaller KD and LT values in the glass surface treatments were probably due to the greater availability of the pyrethroid on the treated glass surface (Chadwick 1985). Such small KD values indi cate that foraging ants would not have enough time to return to the nest and effectivel y spread the toxicant, especially since they have been observed foraging for up to 45 m from the nest (Vail 1996). Fipronil, when applied to glass, exhi bited a longer knock down time for pharaoh ant workers (KD50 value = 194.98 min) and longer lethal time (LT50 value = 496.54 min) than lambda-cyhalothrin. When fipron il was applied to wood, however, there was a drastic increase in lethal time values (LT50 value = 2396.00 min). My results show that only fipronil treated wood exhibited a delay in mortality >24 h. This is consistent with

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41 the speed of kill for effective bait toxicants, wh ich exhibit a delay in mortality equivalent to 1-4 days (Klotz et al. 1996, Klotz et al. 1997a, b, Rust et al. 2004). Although queens do not perform extensive fo raging duties, they often accompany nest relocation events (Sudd 1960). Queens e xposed to lambda-cyhalothrin treated glass and wood exhibited a similar time to death than workers (LT50 values = 164.45 min and 172.13, respectively). This suggests that queens may not require larger doses of lambdacyhalothrin than workers for mortality. Queen s exposed to fipronil treated glass also did not exhibit a strong difference in leth al time values than workers (LT50 values = 521.93 for queens and 496.54 min for workers). Howe ver, queens exposed to fipronil treated wood exhibited a much larger lethal time valu e than workers. This suggests that queens may take much longer to die when e xposed to lower doses of fipronil. Bridge Assay When exposed to a residual treatment, fo raging worker ants are the first members of the colony to be affected. Foraging work ers contact the insectic ide and return to the nest. Nesting workers are the second members of the colony to be affected by a residual treatment. If the foraging workers have e nough time to get back to the nest they may contaminate other workers by nest mate inte ractions, including grooming, trophallaxis, or other social behaviors. Since the amount of insecticide is diluted when passed from ant to ant, the nesting ants may require several doses from multiple foraging ants. If foraging ants have the ability to cross the treatment and return to the nest several times, there would be a greater transferability of the in secticide. A slow-acting insecticide would allow the foraging workers enough time to cross the treatment several times without becoming moribund.

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42 Results from the glass bridge assay show that lambda-cyhalothri n treated glass will successfully kill a large portion of the workers ve ry quickly. This can be attributed to the very fast knockdown and kill of workers. Ov er half of the worker population was killed in 1 d. These affected foragers died before be ing able to spread the toxicant to the nest cell. Consequently, ~20% of the nesting wo rker ants lived through the duration of the test. Lambda-cyhalothrin, when applied to nonporous surfaces such as glass can be effective in quickly killing foraging workers, but should not be relied upon to eliminate all the workers of a colony. Results from the wood bridge assay show ed that lambda-cyhalothrin treated wood killed only a portion of the workers. Afte r 14 d the pyrethroid produced no significant difference in mortality than the control. This is consistent with James et al. (1998) who observed that bands of 0.6% lambda-cyhalothrin applied to tree trunks were ineffective in excluding Argentine ants after 5 wk. When lambda-cyhalothrin was applied to a porous surface, it lost efficacy against foraging work ers. Many of the nesting workers remained unaffected throughout the duration of the test, as evidenced by the lack of high nesting worker mortality. Results from the fipronil treated glass br idge experiment showed that a large portion of the worker population was killed over time. Over 80% of the worker population was killed after 21 d. An 80% reduc tion in foraging ant has been interpreted as providing satisfactory cont rol in field experiments (Rus t et al. 1996, Vega and Rust 2003). However, in my laboratory experiment this reduction in worker population was not sufficient to eliminate the colony.

PAGE 54

43 Results from the fipronil treated wood bridge assay showed that when the toxicant exhibited a delay in toxicity >24 h, there wa s enough time for the toxicant to spread from ant to ant. Fipronil treated wood was also th e only treatment to successfully eliminate all worker ants. Lambda-cyhalothrin was found to be less effective after 14 d in the wood bridge assay. Fipronil, however, was stil l effective in killing fo raging workers through the duration of the test. In order to reach colony elimination, repr oductives should be killed. In order to affect reproductives located in the nest cells, the reproductives must encounter insecticide-contaminated ants. Foraging ants must be able to transfer insecticide to nesting ants, which then transfer insectic ide to the reproductiv es. Both lambdacyhalothrin and fipronil, when applied to glass were ineffective in producing queen mortality. This can be attributed to rapid sp eed of kill. When workers were exposed to treatments on glass, lethal time values we re all <24 h; lambda-cyhalothrin also was ineffective in producing queen mortality due to rapid speed of kill (worker mortality within a few hours). This rapid mortality di d not allow sufficient time for workers to contaminate the nest cell. The only treatment to exhibit significan t queen mortality was wood treated with fipronil. Wood treated with fipronil was also the only treatment to effectively eliminate some of the colonies. This was also the only treatment to exhibit a delay in mortality >24 h. In order to effectively transfer in secticide from worker ants to queens, the insecticide must be slow-acting. Pharaoh ants can start new colonies with just a small amount of brood (Vail 1996). Brood must be affected in order to accomplis h colony elimination. Brood can be affected by residual treatments in several ways. Th e lowered mass of brood at the end of the

PAGE 55

44 study was probably due to the lack of protei n that was brought back to the nest, and subsequent larval cannibalism (Holldobler and Wilson 1990). The mass of brood at the end of the study for the lambda-cyhalothrin tr eated wood was significantly less than that of the control. However, after the 14-day period, the mass of brood increased, suggesting that the pyrethroid was no longe r effective after 14 d. The fo raging workers were able to transverse the treatment, and gather enough protein to ensure larval survival and production. This was also evident in the ex treme reduction in the mass of brood at the end of the experiment Summary Both fipronil and lambda-cyhalothrin eff ectively killed pharaoh ant workers and queens when they are exposed to the treatmen t. No degree of repellency was found for either insecticide. Fipronil and lambda-cyha lothrin exhibited varying times to death of both workers and queens. There was however, an enormous difference in effects of a residual treatment against colonies. Lambda-c yhalothrin was effective in killing a large portion of workers, but did not eliminate th e colony. Fipronil treated wood was the only treatment to result in significant queen mo rtality and a large reduction in brood mass. Some of the colonies in the fipronil applied to wood treatment were eliminated. Fipronil applied to absorbent surfaces, such as wood s hows the greatest potential for management of pharaoh ant infestations.

PAGE 56

45 APPENDIX MANAGEMENT OF INSECT COLONIES Pharaoh ant colonies were rear ed at the University of Fl orida (Gainesville, FL) in a rearing room maintained at 27 1.5C, 40 7% RH. A space heater was operated constantly to maintain a fairly constant te mperature. Colonies were housed in plastic trays (56 by 44 by 12 cm; Panel Controls, Greenv ille, SC) coated on the inner walls with Fluon (AGC Chemicals, Bayonne, NJ) to prevent the ants from escaping. A sheet of clear plastic (60 x 48 cm) was used to cover the trays and maintain a fairly constant relative humidity inside the tray. Four to six ne st cells were placed inside the tray to hold queens, brood, and nesting workers. Cells c onsisted of square plas ter-filled (Dentsply, York, PA) plastic Petri dishes (100 x 15mm; Becton Dickinson Labware, Franklin Lakes, NJ) (Williams 1990). A soldering iron was used to melt a 3 mm hole in the center of each side of the bottom dish to allow the ants access. Yellow acetate paper covered the nest cell to filter li ght and make it more at tractive for brood rearing. Colonies were provided ad libitum with water and 10% sugar water in polypropylene round bottom centrifuge tube s (32 by 164 mm; Fisher Scientific, Pittsburgh, PA) plugged with cotton balls. Freshly killed American cockroaches ( Periplaneta americana), obtained from the Urban Entomology laboratory, University of Florida, Gainesville, FL we re provided in paper cups three times per week. This provided protein for oogenesis and larval development (Chapman 1998). When a new cup of cockroaches was provided, the old cup was shaken to remove foraging ants, and placed into a sealed bag. The bag was frozen for several days before disposal.

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46 Dead ants were removed once per week with an index card. The bottom of the tray was wiped with moist cotton balls to rem ove excrement and regurgitated food matter once per week. All nest cells we re transferred to a clean, Fluon coated tray once every three months. Ants dispersed in the bottom of the old tray were collected on an index card and transferred to the clean tray.

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47 LIST OF REFERENCES Appel, A. G., M. J. Gehret, and M. J. Tanley. 2004. Repellency and toxicity of mint oil granules to red imported fire ants (Hym enoptera: Formicidae). J. Econ. Entomol. 97: 575-580. Arnold, G. 1916. A monograph of the Formicidae of Sout h Africa. Part II. Ponerinae, Dorylinae. Ann. S. Afr. Mus. 14: 159-270. Beekman, M., D. J. T. Sumpter, and F. L. W. Ratnieks. 2001. Phase transition between disordered and ordered foraging in Pharaoh's ants. Proc. Natl. Acad. Sci. 98: 9703-9706. Bolton, B. 1987. A review of the Solenopsis genus-group and revision of the Afrotropical Monomorium Mayr (Hymenoptera: Formicidae). Bull. Brit. Mus. Nat. Hist. (Ent.) 54: 263-452. Bolton, B. 2003. Synopsis and classification of the Formicidae. Mem. Amer. Entomol. Inst. 71: 1-370. Buczkowski, G., M. E. Scharf, C. R. Ratliff, and G. W. Bennet. 2005. Efficacy of simulated barrier treatments against labor atory colonies of ph araoh ant. J. Econ. Entomol. 98: 485-492. Burrus, R. G. 2004. Pharaoh ant consumption of fluids used in hospital environments, M. S. thesis, University of Florida, Gainesville Chadwick, P. R. 1985. Surfaces and other factors m odifying the effectiveness of pyrethroids against insects in public health. Pestic. Sci. 16: 383-391. Chapman, R. F. 1998. The insects. Cambridge University Press, Cambridge, MA. Edwards, J. P. 1986. The biology, economic importance, and control of the Pharaoh's ant, Monomorium pharaonis (L.) (Hymenoptera: Formicidae). pp. 257-271. In S. B. Vinson [ed.], Economic impact and c ontrol of social insects. Praeger Publishers, New York, NY. Edwards, J. P., and L. Abraham. 1990. Changes in food selection by workers of the Pharaoh ant, Monomorium pharaonis. Med. Vet. Entomol. 4: 205-211.

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48 Haack, K. D. 1987. Aspects of the food handling be havior of the Pharaoh ant, Monomorium pharaonis (L.) (Hymenoptera: Formicidae), M. S. thesis, Texas A&M University, Harris, J. R. 1991. Environmental factors affecting th e distribution of established and the formation of satellite co lonies of the Pharaoh ant, Monomorium pharaonis (L.), Ph.D. dissertation, Texas A & M University, Holldobler, B., and E. O. Wilson. 1990. The ants. Belknap Press, Cambridge, Massachusetts. Hooper-Bui, L. M., A. G. Appel, and M. K. Rust. 2003. Preference of food particle size among several urban ant species. J. Econ. Entomol. 95: 1222-1228. Hu, X. P. 2005. Evaluation of efficacy and nonrepel lency of indoxacarb and fiproniltreated soil at various concentrations and thicknesses agai nst subterranean termites (Isoptera: Rhinotermitidae). J. Econ. Entomol. 98: 509-517. Ibrahim, S. A., G. Henderson, and H. Fei. 2003. Toxicity, repellen cy, and horizontal transmission of fipronil in the formos an subterranean termite (Isoptera: Rhinotermitidae). J. Econ. Entomol. 96: 461-467. James, D. G., M. M. Stevens, and K. J. O'Malley. 1998. Prolonged exclusion of foraging ants (Hymenoptera: Formicidae ) from citrus trees using controlledrelease chlorpyrifos trunk bands. Int. J. Pest Manag. 44: 65-69. Jeanson, R., F. L. W. Ratnieks, and J.-L. Deneubourg. 2003. Pheromone trail decay rates on different substrates in the Pharaoh's ant, Monomorium pharaonis Physiol. Entomol. 28: 192-198. Klotz, J. H. 2004. Ants, pp. 635-693. In S. A. Hedges [ed.], Mallis Handbook of Pest Control, 9 ed. Gie Media, Cleveland, OH. Klotz, J. H., and B. L. Reid. 1992. The use of spatial cues for structural guideline orientation in Tapinoma sessile and Camponotus pennsylvanicus (Hymenoptera: Formicidae). J. Insect Behav. 5: 71-82. Klotz, J. H., K. M. Vail, and D. F. Williams. 1997a. Toxicity of a boric acid-sucrose water bait to Solenopsis invicta (Hymenoptera: Formicidae). J. Econ. Entomol. 30: 488-491. Klotz, J. H., K. M. Vail, a nd D. F. Williams. 1997b. Liquid boric acid bait for control of structural infestations of pharaoh ants (Hymenopter a: Formicidae). J. Econ. Entomol. 90: 523-526. Klotz, J. H., L. Greenberg, and E. C. Venn. 1998. Liquid boric acid bait for control of Argentine ant (Hymenoptera: Formic idae). J. Econ. Entomol. 91: 10-914.

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49 Klotz, J. H., D. H. Oi, K. M. Vail, and D. F. Williams. 1996. Laboratory evaluation of a boric acid liquid ba it on colonies of Tapinoma melancephalum, Argentine ants, and Pharaoh ants (Hymenoptera: Formicidae). J. Econ. Entomol. 89: 673-677. Klotz, J. H., L. Greenberg, C. Amrhein, and M. K. Rust. 2000. Toxicity and repellency of borate-sucrose water baits to Argentine ants (Hymenoptera: Formicidae). J. Econ. Entomol. 93: 1256-1258. Knight, R. L., and M. K. Rust. 1990. Repellency and efficacy of insecticides against foraging workers in laboratory colonies of Argentine ants (Hymenoptera: Formicidae). J. Econ. Entomol. 83: 1402-1408. Oi, D. H., K. M. Vail, and D. F. Williams. 1996. Field evaluation of perimeter treatments for Pharaoh ant (Hymenoptera: Formicidae) control. Fl. Entomol. 79: 252-263. Oi, D. H., K. M. Vail, and D. F. Williams. 2000. Bait distribution among multiple colonies of Pharaoh ants (Hymenoptera : Formicidae). J. Econ. Entomol. 93: 1247-1255. Oi, D. H., K. M. Vail, D. F. Williams, and D. N. Bieman. 1994. Indoor and outdoor foraging locations of Pharaoh ants (Hymenoptera: Formicidae) and control strategies using bait stat ions. Fl. Entomol. 77: 85-91. Osbrink, W. L. A., and A. R. Lax. 2002. Effect of tolerance to insecticides on substrate penetration by formosan subterranean termites (Isoptera: Rhinotermitidae). J. Econ. Entomol. 95: 989-1000. Peacock, A. D. 1950. Studies in Pharaoh's ant, Monomorium pharaonis (L.). 4. Eggproduction. Entomol. Mon. Mag. 86: 294-298. Petersen-Braun, M. 1975. Investigations on the social or ganization in the Pharaoh's ant, Monomorium pharaonis (L.) (Hymenoptera: Formicidae). I. The regulation of the brood cycle. Insectes Soc. 22: 269-291. Pranschke, A. M., L. M. Hooper-bui, and B. Moser. 2003. Efficacy of bifenthrin treatment zones against red imported fire ant. J. Econ. Entomol. 96: 98-105. Ritter, F. J., I. E. M. Bruggemann-Rotgans, P. E. J. Verwiel, C. J. Persoons, and E. Talman. 1977. Trail pheromone of the pharaoh's ant, Monomorium pharaonis: isolation and identification of faranal, a terpenoid related to juvenile hormone. Tetrahedron Lett. 30: 2617-18. Rust, M. K., K. Haagsma, and D. A. Reierson. 1996. Barrier sprays to control Argentine ants (Hymenoptera: Form icidae). J. Econ. Entomol. 89: 134-137.

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50 Rust, M. K., D. A. Reierson, and J. H. Klotz. 2004. Delayed toxicity as a critical factor in the efficacy of aqueous baits for c ontrolling Argentine ants (Hymenoptera: Formicidae). J. Econ. Entomol. 97: 1017-1024. Scharf, M. E., C. R. Ratliff, and G. W. Bennet. 2004. Impacts of resi dual insecticide barriers on perimeter-invading ants, with particular reference to odorous house ant, Tapinoma sessile J. Econ. Entomol. 97: 601-605. Silverman, J., and T. H. Roulston. 2001. Acceptance and intake of gel and liquid sucrose compositions by the Argentine an t (Hymenoptera: Formicidae). J. Econ. Entomol. 94: 511-515. Silverman, J., and T. H. Roulston. 2003. Retrieval of granular bait by the Argentine ant (Hymenoptera: Formicidae): Effect of clumped versus scattered dispersion patterns. J. Econ. Entomol. 96: 871-874. Soeprono, A. M., and M. K. Rust. 2004a. Effect of horizontal transfer of barrier insecticides to control Argentine ants (Hymenoptera: Formicidae). J. Econ. Entomol. 97: 1675-1681. Soeprono, A. M., and M. K. Rust. 2004b. Effect of delayed toxicity of chemical barriers to control Argentine ants (Hymenoptera: Formicidae). J. Econ. Entomol. 97: 2021-2028. Statistical Analysis Softw are Institute (SAS). 2002. Statistical analysis software computer program computer program, vers ion 8.01. Institute, S. A. S., Cary, NC. Stringer, J., C. E., C. S. Lofgren, and F. J. Bartlett. 1964. Imported fire ant toxic bait studies: Evaluation of toxicants. J. Econ. Entomol. 57: 941-945. Sudd, J. H. 1960. The foraging method of Pharaoh's ant, Monomorium pharaonis (L.). Anim. Behav. 8: 67-75. Throne, J. E., D. K. Weaver, V. Chew, and J. E. Baker. 1995. Probit analysis of correlated data: multiple observations ove r time at one concentration. J. Econ. Entomol. 88: 1510-1512. Vail, K. M. 1996. Foraging, spatial distribution, and control of the Pharaoh ant, Monomorium pharaonis (L.), Ph.D. dissertation, University of Florida, Gainesville Vail, K. M. 2002. Management of structure invadi ng ants, University of Tennessee Extension Bulletin 1629. Vail, K. M., and D. F. Williams. 1994. Foraging of the Pharaoh ant, Monomorium pharaonis: an exotic in the urban environment. In D. F. Williams [ed.], Exotic Ants: Biology, Impact, and Control of Introduced Species. Westview Press, Boulder, CO.

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51 Vail, K. M., D. F. Williams, and D. H. Oi. 1996. Perimeter treatments with two bait formulations of pyriproxyfen for cont rol of pharaoh ants (Hymenoptera: Formicidae). J. Econ. Entomol. 89: 1501-1507. Vega, S. Y., and M. K. Rust. 2003. Determining the foraging range and origin of resurgence after treatment of Argentine an t (Hymenoptera: Formicidae) in urban areas. J. Econ. Entomol. 96: 844-849. Williams, D. F. 1990. Effects of fenoxycarb baits on la boratory colonies of Pharaoh's ant, Monomorium pharaonis pp. 676-683. In R. K. e. a. Vander Meer [ed.], Applied myrmecology: a world pers pective. Westview, Boulder, CO. Wilson, E. O. 1971. The insect societies. Harvard University Press, Cambridge, MA.

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52 BIOGRAPHICAL SKETCH David Melius was born on February 1 2, 1982, in Kingston, NY, to Steven and Donna Melius. He has two sisters, Maria a nd Gina Melius. He and his family spent 15 years in Hurley, NY. In the summer of 1995, his father was transferred to Boca Raton, FL. David attended Olympic Heights High School from 1995-2000. After high school, David attended the University of Florid a starting in August of 2000. He earned a Bachelor of Science degree in entomo logy and nematology in December of 2003. While earning his Bachelor of Scien ce degree, David began working as a laboratory technician for Dr. Phil Koehler in October of 2002. He gained experience with rearing urban pest insect s and managing insecticide field research. Such experience captured his interest in graduate school and urban pest insects. He remained at the University of Florida where he completed a master’s degree in urban entomology.


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Physical Description: Mixed Material
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REPELLENCY, LETHAL TIME, AND TRANSFERENCE OF RESIDUAL
INSECTICIDES USED FOR PHARAOH ANT CONTROL















By

DAVID ANTHONY MELIUS


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

David Anthony Melius

































This thesis is dedicated to my mom, Donna Melius.















ACKNOWLEDGMENTS

I would like to thank my parents and sisters for their continuous support. I would

like to thank my friends including Colin Hickey, Justin Saunders, and Ryan Welch.

I greatly appreciate the efforts of Tiny Willis and Gil Marshall in helping to

gather supplies for all of my experiments, as well as for their constant help and

generosity. I also would like to thank Paul Kaiser for his help in setting up, recording

data, and taking down experiments.

I would like to thank Debbie Hall and Josh Crews for their consistent problem

solving skills. I would also like to thank Nancy Sanders for her kindness, support, and

encouragement. I greatly appreciate the efforts of Jane Medley in preparing extension

poster and booklet publications with me and other students in the urban lab.

I would like to thank my advisor, Dr. Phil Koehler, for both his financial support

as well as continual help with statistics and experimental design. He has also provided

many opportunities for developing presentations. These involved many different

audiences including pest control operators, pesticide company technicians, master

gardeners, and other entomologists. These presentations have greatly helped me hone my

knowledge on urban pests and entomology.

I greatly appreciate Dr. Richard Patterson, who has always been extremely

accommodating. I would also like to thank Dr. David Williams for his constant help and

advice. He has always been available as soon as I need help. He has a vast knowledge of

ant biology, control, and experimentation that he was always willing to share with me.

iv









Finally, I would like to thank my girlfriend, Kendra Pesko. I greatly appreciate her

advice and editorial skills. She has helped me get through both good times as well as

bad.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ........ .................................................. ....... ........ viii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

A B ST R A C T ................. .......................................................................................... x

CHAPTER

1 IN TRODU CTION ................................................. ...... .................

2 LITER A TU R E R EV IEW .............................................................. ....................... 4

O rigin and D distribution .................................................................. .......................4
Food and Foraging ....................................................... .......... ............. ....
M medical Importance .................. .................. ........................ .. .......... 7
C o n tro l ............................................................................ . 8

3 M ATERIALS AND M ETHOD S ........................................ ......................... 13

In se c ts ................................................................................1 3
Insecticide Treatm ents. ........................................... ................ .... ... .... 13
Repellency A ssay .................................... ........... .......... .... 14
L ethal T im e A ssay .............................................. .. ................ ............. ..... 14
B rid g e A ssay ......................................................................................................... 1 5
D ata A n a ly sis ............................................................................................1 6

4 R E S U L T S .............................................................................2 0

R epellency A ssay.....................................................................................20
Lethal Tim e A ssay ..........................................................................20
B rid g e A ssay ......................................................................................................... 2 1

5 D ISC U S SIO N ............................................................................... 37

F o re w o rd .......................................................................................................3 7
R epellency A ssay.....................................................................................38
Lethal Tim e A ssay ................................. .................................. 40









B rid g e A ssay ......................................................................................................... 4 1
S u m m a ry ............................................................................................................... 4 4

APPENDIX MANAGEMENT OF INSECT COLONIES ...........................................45

L IST O F R E F E R E N C E S ........................................................................ .....................47

B IO G R A PH IC A L SK E TCH ..................................................................... ..................52
















LIST OF TABLES


Table p

4-1 Distribution of 50 worker ants in release, untreated, and water, fipronil, or
lambda-cyhalothrin treated Petri dishes ....................................... ............... 24

4-2 Repellency of fipronil or lambda-cyhalothrin treated glass to pharaoh ant
w ork ers ........................... .................................... .. .................. 2 5

4-3 KD values of pharaoh ant workers when exposed to glass or wood treated with
fipronil or lam bda-cyhalothrin ........................................... .......................... 26

4-4 LT values of pharaoh ant workers and queens when exposed to glass or wood
treated with fipronil or lambda-cyhalothrin .................................. ............... 27

4-5 Percent mortality of pharaoh ant workers in response to placement of glass or
wood bridges treated with water, fipronil, or lambda-cyhalothrin.........................28

4-6 Percent mortality of pharaoh ant queens in response to placement of glass or
wood bridges treated with water, fipronil, or lambda-cyhalothrin.........................29

4-7 Mass of brood before and after placement of bridges treated with fipronil or
lam bda-cyhalothrin........................... ..... .......................... .......... 30
















LIST OF FIGURES


Figure page

3-1 Repellency assay setup showing location of release, treated, and untreated
dishes .......... .... ........................................... ............ ........ 17

3-2 Bridge assay setup showing location of nest cell, bridge, and food sources............18

3-3 Glass bridge assay setup showing ants trailing along bridge ...............................19

4-1 Series of photographs of Control (replicate 1) showing nest cell over time............31

4-2 Series of photographs of Control (replicate 2) showing nest cell over time............32

4-3 Series of photographs of lambda-cyhalothrin (replicate 1) showing the nest cell
over tim e .................................. .................................. ......... 33

4-4 Series of photographs of lambda-cyhalothrin (replicate 2) showing nest cell over
tim e ............................................................................. 3 4

4-5 Series of photographs of fipronil (replicate 4) showing nest cell over time ............35

4-6 Series of photographs of fipronil (replicate 5) showing nest cell over time ............36















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

REPELLENCY, LETHAL TIME, AND TRANSFERENCE OF RESIDUAL
INSECTICIDES USED FOR PHARAOH ANT CONTROL

By

David Anthony Melius

December 2005

Chair: Philip Koehler
Major Department: Entomology and Nematology

Pharaoh ants, Monomorium pharaonis (L.), are nuisance pests, which have

polygynous, polydomous colonies, and reproduce by budding, making control very

difficult. Baiting is generally believed to be the only control method that results in

pharaoh ant colony elimination. Effective baits should not be repellent, possess a slow

acting toxicant, and be readily transferred between ants. If a residual treatment could

mimic the characteristics of an effective bait toxicant, it could be an effective treatment

for pharaoh ants. Fipronil and lambda-cyhalothrin were evaluated as residual treatments

for infestations of the pharaoh ant.

Both fipronil and lambda-cyhalothrin effectively killed pharaoh ant workers and

queens when they were exposed to the treatment. No degree of repellency was found for

either insecticide. Glass and wood panels were used to estimate knock down and lethal

time values of pharaoh ant workers and queens. Lambda-cyhalothrin exhibited low LT50

values for workers on both glass and wood surfaces tested (LT5o values = 137.84 and









158.24 min for glass and wood respectively), indicating that foraging pharaoh ant

workers would have very little time to return to the nest. Fipronil treated glass also

resulted in a fairly low lethal time value (LT5o = 496.54 min). A delay in mortality >24 h

for pharaoh ant workers was only evident in the fipronil treated wood group (LT5o =

2396.00 min), indicating that foraging workers would have time to return to the nest.

Similar results were found for pharaoh ant queens exposed to fipronil and lambda-

cyhalothrin.

A bridge experiment was conducted in order to determine if a residual treatment of

fipronil or lambda-cyhalothrin could be effectively transferred from foraging workers to

affect the rest of the colony. Glass treatments for both insecticides were effective in

eliminating -80% of the worker population. However, no significant queen mortality

was evident throughout the test. Wood bridges treated with lambda-cyhalothrin appeared

to lose effectiveness after 2 wk and only resulted in 63% worker ant mortality after 21 d.

Fipronil treated wood was the most effective treatment, with 100% worker mortality,

80% queen mortality, and a heavy reduction in brood after 21 d.

A delay in mortality >24 h, and preferably in the range of 3-5 days was crucial for

effective transference of insecticides throughout the colony. This was only evident in

wood treated with fipronil. Some of the colonies in the fipronil applied to wood

treatment were eliminated. Fipronil residual treatments applied to absorbent surfaces,

such as wood, showed the greatest potential for management of pharaoh ant infestations.














CHAPTER 1
INTRODUCTION

Pharaoh ants, Monomorium pharaonis (L.), are nuisance pests, which have

polygynous, polydomous colonies, and generate new colonies by budding. Budding

involves the carrying of brood by workers to a new nesting location. Queens are not

necessary for the formation of a new satellite nesting location but will sometimes

accompany the movement (Edwards 1986). Ants often share resources between these

satellite nests (Holldobler and Wilson 1990). This budding behavior has aided the

pharaoh ant in becoming an intense household pest. The slightest disturbance, including

spraying repellent insecticides, can lead to the formation of new satellite nests, making it

very difficult for pest control operators to manage pharaoh ant infestations (Holldobler

and Wilson 1990, Buczkowski et al. 2005).

Colonies typically employ only a small percentage of workers to serve as foragers

(Williams 1990, Vail 1996). This behavior also makes management extremely difficult.

Control measures must also include methods of eliminating the reproductive caste of the

colonies. Direct treatment of all nesting locations would be an ideal control stratagem.

However, pharaoh ants primarily nest indoors, including inside wall voids, in cabinets

and under sinks (Edwards 1986). These inaccessible nesting locations, coupled with the

polydomous behavior of the pharaoh ant, make direct treatment of all nests nearly

impossible.

Management of pharaoh ant infestations includes the use of baits and residual

sprays. Effective baits should possess a slow acting toxicant, be readily transferred









between ants, and should not be repellent (Stringer et al. 1964). The bait toxicant should

possess all three characteristics or it will not be effective in eliminating an infestation.

Baiting can be very effective, but is also time consuming and can take up to 8-12 weeks

to reach control (Klotz et al. 1996, Oi et al. 2000). Pharaoh ants exhibit changes in food

preference depending upon satiation of one food source (Edwards and Abraham 1990).

This makes it difficult to control infestations with the use of a single bait formulation.

The bait should also be available for a long time interval, and the ants must feed on it for

at least 3 d (Klotz et al. 1997b).

Residual insecticides provide pest control operators with another treatment option.

There are different treatment methods for residual insecticides depending upon the

species of ant that is involved. Perimeter-invading ants, such as the Argentine ant and

red imported fire ant can be excluded from a structure with the application of a barrier of

repellent or nonrepellent insecticides. Knight and Rust (1990) evaluated residual

insecticides against laboratory Argentine ant workers and found that insecticides high in

repellency or low in repellency can still be an effective barrier treatment. Bifenthrin

barrier treatments were 100% effective in excluding red imported fire ants from

structures up to 15 wk after treatment (Pranschke et al. 2003). Recently, perimeter

treatments with fipronil dramatically reduced foraging activity of structure invading ants

(Scharf et al. 2004). Since pharaoh ants are interior nesting ants, the use of a repellent

perimeter treatment would trap the ants inside the structure and not be an effective

control measure. The use of perimeter treatments for control of pharaoh ants relies on the

ants crossing the treatment zone. Although pharaoh ants nest indoors, Oi et al. (1994)









found that in Florida, most of the foraging activity occurs outdoors. Placement of bait

stations outdoors can be an effective treatment (Oi et al. 1994, Oi et al. 1996).

If a residual treatment could mimic the characteristics of a bait toxicant, it could be

an effective treatment strategy for pharaoh ant infestations. The residual toxicant should

not cause foraging ants to avoid the treated surface, it should have a delay in toxicity so

the foraging ants could effectively spread the toxicant once they reach the nest, and it

should be transferable between ants via contact or trophallaxis. Such a treatment could

allow pest control operators to manage a pharaoh ant infestation in a much more efficient

manner.

The objective of this study was to determine the efficacy of a residual treatment

against infestations of the pharaoh ant. A repellency experiment was conducted in order

to determine if foraging ants would walk on treated surfaces. An effective treatment

should not repel foraging ants from contacting a treated surface. A lethal time

experiment was conducted on both pharaoh ant workers and queens in order to determine

the time until death after exposure to the treatment. The treatment should exhibit a delay

in mortality, where death would occur in 1-5 d. Finally, a bridge experiment was

conducted in order to determine effects of a residual treatment on workers and

reproductive within the colony. Residual treatments should affect not only foraging

workers but also nesting workers and reproductive located within the nest.














CHAPTER 2
LITERATURE REVIEW

Origin and Distribution

The African-Middle Eastern region is thought to be the place of origin of this ant

(Arnold 1916, Bolton 1987). Through commerce, it has become worldwide in

distribution (Edwards 1986). Artificial heating has allowed this once tropical ant to

spread to temperate regions (Harris 1991).

Biology

M. pharaonis belongs to the subfamily Myrmicinae, within the family Formicidae,

and the tribe Solenopsidini (Bolton 2003) The workers ofM. pharaonis are yellowish

brown to reddish and reach approximately 2 mm in length (Vail 1996). They possess 2

nodes, and a 12-segmented antennae with a 3-segmented club. The queens of this species

are roughly twice the size of workers (4 mm) and possess a definitively larger gaster than

the workers (Haack 1987). Males are black and possess wings.

Colonies are polygynous. Several hundred queens can reside in one nest. M.

pharaonis is closely associated with human habitation and is rarely found nesting

outdoors (Vail 1996). Any warm area with high humidity can be utilized as a nesting

location (Edwards 1986). Thus, wall voids, attics, door housings, electrical outlets, sinks,

and other crevices within the home serve as nesting sites (Vail 1996). Colonies are also

polydomous, possessing multiple nesting locations, with little or no aggression between

nests (Edwards 1986, Haack 1987).









Colony foundation does not occur through a single fertilized female, as in most ant

species. Instead, reproduction occurs by sociotomy (budding). Workers will carry brood

to a separate nesting location. Queens are not necessary for the survival of the new nest,

however, they will often accompany the workers (Edwards 1986). Migrating workers

may set up temporary nesting locations before settling at a final site. These temporary, or

satellite, nests may be connected by odor trails and may represent a single, enormous

interconnected colony (Harris 1991). Several conditions can instigate sociotomy

including overcrowding, environmental conditions, and lack of food or water (Edwards

1986). Recent evidence suggests that the application of repellent insecticides can also

provoke sociotomy (Buczkowski et al. 2005).

Although, newly ecdysed, virgin queens temporarily possess wings, they are

incapable of flight. Males will mate with the females from the same, or a nearby colony

on the ground. This lack of outbreeding has led to a lack of aggression between nests,

and has helped develop the behavior of budding (Wilson 1971). Oviposition occurs

quickly after mating. In the first 4-5 weeks, the queen will lay 4-6 eggs per day. The rate

of oviposition then rises to 25-35 eggs per day. During the last 5 weeks of the queen's

life, the oviposition rate drops to 3-7 eggs per day (Edwards 1986). The maximum

lifespan of the queen has been measured at 39 weeks in the field (Peacock 1950) and over

52 weeks in the laboratory (Edwards 1986). The average lifespan of a queen is about 200

days (Petersen-Braun 1975). Over her lifespan, a queen may lay over 4500 eggs

(Edwards 1986). Such reproductive potential allows for the quick development of large

colonies, despite high larval mortality (Peacock 1950). Brood is separated into different

stages of development probably to curb larval cannibalism (Edwards 1986).









Peacock recorded the following development times for the worker caste: egg 7.3

days, larvae 17 days, prepupa 3.1 days, pupa 9 days. The average life cycle is completed

at 36.4 days. The life cycle of the reproductive caste is completed at 41.25 days (1950).

Subsequent studies by Petersen-Braun (1973), Kretzchmar (1973), and Berndt and

Eichler (1987) have given similar results (Vail 1996).

The worker caste exhibits age polyethism. The younger workers will remain in the

nest and tend the brood. The older workers forage for food and water (Holldobler and

Wilson 1990). Worker ants live about 9-10 weeks. The older workers constitute a small

fraction (0.7 to 5.6%) of the colony due to their short lifespan (Vail 1996).

Food and Foraging

M. pharaonis are omnivorous and will feed on various sources of lipids,

carbohydrates, and proteins (Edwards 1986). The larvae and queen of the colony require

large amounts of protein for development and vitellogenesis (Chapman 1998). Dead

insects found at lights and window sills are probably the most important source of this

protein (Sudd 1960, Oi et al. 1994). Foraging workers transport protein back to the nest

and feed it to the larvae. The larvae may help the adult workers receive nutrients that

they cannot consume. Stomodeal as well as proctodeal trophallaxis from larvae to

worker has been documented (Holldobler and Wilson 1990). The salivary and rectal

fluid extracted from the larvae ofM. pharaonis have been shown to contain nitrogen,

amino acids, proteases, and carbohydrases (Holldobler and Wilson 1990). These

compounds are necessary for the breakdown of worker food sources as well as the

production of additional enzymes. Edwards and Abraham showed that M. pharaonis

exhibits a satiation and alternation response to different food sources. When presented

with a highly attractive food for several weeks, foraging ants will consume an alternative









food source when given a choice (1990). This behavior allows the colony to receive a

balanced diet.

Worker ants display various foraging behaviors. Initial foraging consists of worker

ants wandering in search of food. Foraging ants will often orient along a structural

guideline (Klotz and Reid 1992). Upon finding food, the worker will return to the nest in

the straightest possible path (Klotz 2004). While returning, the ant will lay a trail

pheromone, which will lead additional workers straight to the food source. The trail

pheromone has been identified as faranal, (6E, 10Z)-3,4,7,11-tetramethyl-6,10-

tetradecadienal (Ritter et al. 1977, Holldobler and Wilson 1990). Beekman et al. (2001)

showed that the use of trail pheromones can only be sustained in large colonies (over 600

ants). Smaller colonies cannot sustain ordered foraging behavior, possibly due to the

ephemeral nature of the trail pheromone. Jeanson et al. (2003) showed that the trail

pheromone decays in 10 minutes on a plastic surface. If additional ants do not reinforce

the trail, it will disappear. Smaller colonies will typically forage in a random wandering

fashion.

Medical Importance

For many years, the importance ofM. pharaonis has been centralized on its pest

status. Potential for disease transmission has increased the importance of this ant. In

1972, worker ants were shown to transport Pseudomonas, Staphylococcus, Salmonella,

Clostridium, and Streptococcus (Beatson). Additionally, the pharaoh ant has been shown

to vector Bordetella bronchiseptica, a swine pneumonia (Beatson 1972). Hospitals,

including intensive care units have been infested. Recently, Burrus (2004) showed that

pharaoh ants will readily consume many fluids found in hospital environments, including

dextrose, Ensure, plasma, and saline.









Control

Pharaoh ants are among the most difficult pests to manage effectively. Their social

status makes them especially cumbersome for both homeowners as well as pest control

operators. Several factors account for this including their polydomous nesting behavior

and polygyny. The most effective method of eliminating an ant infestation is to treat the

nest directly. However, this becomes extremely difficult when the ants have multiple

nesting locations which are linked and share resources (Vail 1996). Since pharaoh ant

colonies typically have several hundred queens, management efforts must include

methods of delivering insecticide to the reproductive.

Management of pharaoh ant infestations must include identification, monitoring,

and implementation of an integrated pest management program. Proper identification is

essential. Pharaoh ants are very similar in appearance to fire ants as well as other

perimeter-invading ants. If pharaoh ants were misidentified as a perimeter-invading ant,

effectively eliminating an infestation would be difficult. Proper monitoring can help to

ensure that treatment methods are working properly. Integrated pest management

methods include exclusion, physical removal, sanitation, and chemical management.

Exclusion is important for keeping ants from entering a structure. Cracks and

crevices should be properly sealed. Window screens should be free of tears. Although

this will not remove an existing infestation, it may help prevent ants from colonizing

inside.

Sanitation is crucial for proper management of ant infestations. Ants are extremely

susceptible to dehydration. Leaky faucets must be fixed and extraneous sources of water

must be eliminated. Crumbs and food spills should be cleaned immediately. Without

proper sanitation, ant infestations cannot efficiently be managed.









Chemical management methods for pharaoh ant control have employed the use of

residual sprays and toxic baits. In the past, repellent residual sprays have been used.

However, such compounds will only kill the foraging workers (Williams 1990, Vail

1996). This represents only 0.7 to 5.6% of the entire colony (Vail 1996). Repellent

sprays may also lead to the formation of satellite nests (Vail and Williams 1994, Vail

2002, Buczkowski et al. 2005). Thus, repellent compounds may actually promote

dispersion and growth of a pharaoh ant infestation. Colonies ofM. pharaonis are able to

resist starvation for prolonged periods of time with the aid of larval trophallaxis

(Holldobler and Wilson 1990). Thus, even if a repellent spray were to cut a colony off

from its food source, it could still survive for long periods of time.

It is generally believed that baits are the only control method that will achieve

complete eradication of pharaoh ant infestations (Klotz et al. 1996, Klotz 2004). Baiting

takes advantage of the fact that ants will forage and recruit to food sources. Bait is placed

in cracks, crevices, along active trails, and other possible ant congregation areas. The

foraging ants feed on the bait and transmit the toxicant to nest mates via trophallaxis.

Baits employ the use of insect growth regulators or stomach poisons (Vail 1996).

Formulations for baits include granular, gel, liquid, and stations. Hooper-Bui et al.

(2003) found that pharaoh ants prefer granule sizes between 420-590 micrometers. Most

granular bait particles are 1,000 to 2,000 micrometers, however, Niban particles are less

than 420 micrometers (Hooper-Bui et al. 2003). Current baits include the following:

Bayer Maxforce Ant Killer Granular bait (Hydramethylnon), Niban Puffer Fine Granular

Bait (Orthoboric acid), Whitmire Micro-gen Advance Ant Bait (Abamectin). When









applying a granular bait, it was found that dispersion pattern is not important (Silverman

and Roulston 2003).

Gel baits are a very popular formulation with pest control operators, however, it

was found that consumption of liquid bait is higher than gel formulated bait in Argentine

ants (Silverman and Roulston 2001). Although gel baits are more convenient for

transport, application, and longevity, they may not be as effective as a liquid formulation

(Silverman and Roulston 2001). Current gel baits include: Gourmet Ant Bait

(Disodium octaborate tetrahydrate), Bayer Maxforce FC Ant Killer Bait Gel (Fipronil),

Intice Sweet Ant Gel (Orthoboric acid), and Drax Ant Bait (boric acid).

Liquid boric acid baits have been evaluated against colonies of ants many times

(Klotz et al. 1996, Klotz et al. 1997a, b, Klotz et al. 1998, Klotz et al. 2000, Rust et al.

2004). Liquid baits are generally found to reduce the worker population and foraging

activity after 2-8 wks. Commercial liquid baits include Whitmire Micro-Gen Advance

Liquid Ant Bait (boric acid), Terro Ant Killer II (Disodium octaborate tetrahydrate), and

Uncle Albert's Liquid ant bait (boric acid).

Baits are also formulated as bait stations. Bait stations often employ liquid or gel

bait within a plastic container. This method keeps the toxicant out of reach of children

and pets. Current bait stations include: FMC Fluorguard Ant Bait Stations

(Sulfluramid), Bayer Maxforce Ant Bait Stations (Hydramethylnon), Whitmire Micro-

gen DualChoice Ant Bait Stations (Sulfluramid), and Raid Max Ant Bait.

Oi et al. (2000) showed that insect growth regulator baits, although slower acting,

allow for greater transfer of the toxicant among the colony. Currently, Pharorid









(methoprene) is an insect growth regulator labeled for pharaoh ant infestations. Pharorid

is not formulated as a pre-mixed bait, and can be combined with any type of food source.

Although pharaoh ants nest indoors, it was found that in Florida, most of the

foraging activity occurs outdoors (Oi et al. 1994). Placement of bait stations outdoors

can be an extremely effective treatment (Oi et al. 1994, Oi et al. 1996, Vail et al. 1996).

If infestations could be eliminated with an outdoor treatment, both pest control operators

as well as homeowners would benefit. The pest control operator could evaluate and treat

the structure without intruding or placing chemicals within the home.

The use of perimeter treatments for control of ant infestations has been evaluated

many times (Knight and Rust 1990, Oi et al. 1994, Oi et al. 1996, Rust et al. 1996, Vail et

al. 1996, Pranschke et al. 2003, Scharf et al. 2004, Soeprono and Rust 2004a, Soeprono

and Rust 2004b, Buczkowski et al. 2005). Perimeter treatments with residual insecticides

have become an important tool for pest control operators. Bifenthrin barrier treatments

have been shown to be 100% effective in excluding red imported fire ants from structures

up to 15 wk after treatment (Pranschke et al. 2003). Knight and Rust (1990) evaluated

several residual insecticides against laboratory Argentine ant workers for repellency and

efficacy. Their results indicated that efficacy was not necessarily dependant upon

repellency. When workers encountered less repellent compounds, they were more likely

to receive a higher dose of the insecticide.

Nonrepellent insecticides have increased the popularity of perimeter treatments.

Such compounds rely on the ants' inability to detect the presence of the chemical. The

ants walk through the treatment zone, and eventually gather enough toxicant and die. In

the process, the toxicant is transferred to nestmates through social grooming, trophallaxis,









necrophoresis, and other nestmate interactions. Such horizontal transfer of insecticide

greatly increases the effectiveness of a perimeter treatment (Soeprono and Rust 2004a).

Recently, perimeter treatments with fipronil have been shown to dramatically affect

foraging activity of structure invading ants (Scharf et al. 2004). Much like bait toxicants,

fipronil can be horizontally transferred between nest mates, thus increasing its

effectiveness (Soeprono and Rust 2004a).

The efficacy of a residual treatment for management of an ant infestation is

dependant upon many factors, including, the type of surface the treatment is applied to,

nesting location of the ants, formulation of the insecticide, transferability of the

compound, and delay in toxicity of the compound (Chadwick 1985, Knight and Rust

1990, Osbrink and Lax 2002, Soeprono and Rust 2004a, Soeprono and Rust 2004b).














CHAPTER 3
MATERIALS AND METHODS

Insects

Pharaoh ant colonies were reared at the University of Florida (Gainesville, FL) in a

rearing room maintained at 27 1.50C, 40 7% RH. Colonies were provided ad libitum

with water, 10% sugar water, and freshly killed American cockroaches (Periplaneta

americana), obtained from the Urban Entomology laboratory, University of Florida,

Gainesville, FL). Plastic trays (56 by 44 by 12 cm; Panel Controls, Greenville, SC)

coated on the inner walls with Fluon (AGC Chemicals, Bayonne, NJ) served as rearing

containers. Nest cells consisted of square plaster-filled (Dentsply, York, PA) plastic Petri

dishes (100 x 15mm; Becton Dickinson Labware, Franklin Lakes, NJ)(Williams 1990).

A soldering iron was used to melt a 3 mm hole in the center of each side of the bottom

dish to allow the ants access. Yellow acetate paper covered the nest cell to filter light and

make it more attractive for brood rearing.

Insecticide Treatments.

Treatments consisted of fipronil (Termidor SC 9.1% [AI]; BASF Research Triangle

Park, NC), lambda-cyhalothrin (Demand CS 9.7% [AI]; Syngenta, Wilmington, DE), and

deionized water as a control. Insecticides were mixed resulting in solutions equivalent to

the manufacturer's label rates for ant control (0.06% for fipronil and 0.015% for lambda-

cyhalothrin). Testing areas were placed within a 929 cm2 paper sheet and 3.78 ml of

solution was applied with an airbrush (Paasche, Type H, Chicago, IL) to the entire

surface of the sheet. All testing areas were allowed to dry for 24 h prior to use.









Repellency Assay

Arenas consisted of three Fluon rimmed polystyrene Petri dishes (100 by 15 mm;

Fisher Scientific, Pittsburgh, PA) connected with a wire mesh bridge (0.64 cm mesh, 23

gauge; LG Sourcing, North Wilkesboro, NC). The wire mesh was cut into a 'Y' shape

(15 by 10 cm) and bent to allow the ants access from the untreated release dish to any of

the arenas. One of the arenas was treated with deionized water while the other was

treated with either fipronil or lambda-cyhalothrin. Mortality and location of live ants

were recorded in each arena every 15 min for 1 h. Repellency was defined as the mean

percentage of live ants in the untreated arena (Appel et al. 2004). Six replicates were

performed for each treatment with fifty ants in each replicate.

Lethal Time Assay

Treatments were applied to glass (297 by 50 by 3 mm) or painted plywood panels

(297 by 50 by 10 mm; White Flat Latex Exterior; Masco, Santa Ana, CA). Four Fluon

coated CPVC pipe pieces (40 by 40 cm) were hot-glued to the midline of each panel at 5

cm increments. Ten worker and ten queen ants were placed in each pipe piece. Mortality

and knockdown were recorded every 15 min for 2 h and every 30 min thereafter for 12 h

or until all ants were dead for up to 5 d. In cases where ants were still alive at 12 h,

observations were suspended and resumed 24 h after treatment. Recording of control

mortality ceased when the last ants in the treatments died. Knockdown was defined as

the inability of the ant to right itself, and mortality was defined as having no response

when probed. Six replications per panel type per treatment type were conducted for a

total of 36 experimental units.









Bridge Assay

Experimental colonies consisted of 1000 worker ants (500 selected from outside

nest cells and 500 selected from inside nest cells), 12 queens, and 200 mg of brood held

in Fluon coated plastic trays (56 by 44 by 12 cm; Panel Controls, Greenville, SC). The

500 outside worker ants were placed directly in the tray, while queens, brood, and 500

inside ants were placed into plaster-filled polystyrene Petri dishes (60 by 15 mm; Becton

Dickinson Labware, Franklin Lakes, NJ) which served as nest cells. Nest cells were

covered with yellow acetate paper and placed at one end of the tray. Water and 10%

sugar water were provided ad libitum. Experimental colonies were allowed to acclimate

for 24 h. After the 24 h acclimation period, a deli cup, (237 ml; Fabri-Kal, Kalamazoo,

MI) which had been coated on both vertical sides with Fluon, was centered at the

opposite end of the tray from the nest cell, 7 cm from the front. A wood support (6 by 5

cm) was placed inside the Fluon coated cup. A second wood support was placed near

the nest cell, 197 mm from the first support (see Fig. 2-2). Treated glass (297 by 50 by 3

mm) or treated painted plywood bridges (297 by 50 by 10 mm) were placed on the two

wood supports. The Fluon coated cup prevented ants from entering or exiting the cup

without crossing the bridge (see Fig. 2-3). To provide food and induce foraging, a dead

adult cockroach was placed inside the deli cup every other day throughout the duration of

the experiment. Worker and queen mortality was recorded at 1 h, and at 1, 3, 7, 14, and

21 d. Moribund (unable to right themselves) ants were considered dead. Dead ants were

removed at the time of counting. Digital photographs were taken of the nest cell at each

observation period prior to removal of dead ants for the wood bridge assay (Sony Cyber-

shot model number DSC-W1; Sony, Pittsburg, PA). Additionally, brood was weighed









prior to treatment and 28 d after placement of bridges. Each treatment was replicated six

times per surface type.

Data Analysis

Numbers of live and dead ants in each of the three Petri dishes were analyzed by

one-way analysis of variance, with means separated by Student Newman-Keuls test when

P values of the analysis of variance were significant. Percentage of responding ants that

were located in the treated dish was calculated for each treatment. A paired one-tailed t-

test (a= 0.05, SAS Institute 2002) was used to determine whether the percentage of

responding ants differed significantly from 50% (Ho: Percentage 50% = 0). For the

lethal time assay KD50 and LT50 values were estimated by probit analysis of correlated

data: multiple observations at one concentration (Throne et al. 1995), SAS Institute

2002). Significant differences were determined by nonoverlap of the 95% confidence

intervals (CI). For the bridge assay, cumulative worker and queen mortalities for each

treatment were analyzed with a one-way analysis of variance at each time after treatment,

and means were separated using Student Newman-Keuls test when P values for the

analysis of variance were significant (a= 0.05, SAS Institute 2002).







































Figure 3-1. Repellency assay setup showing location of release, treated, and untreated
dishes. A 'Y' shaped bridge of steel mesh was used to connect all three
dishes. The inner rims of all dishes were coated with Fluon to prevent ant
escape. 50 ants were placed into release dish and allowed to move to any of
the three dishes.














Nest cell
(60 mm diameter;
15 mm deep)


Water vials
(15 ml)



w


Sugar water
vials (15 ml)


Fluon coated
cup (237 ml)


44 cm


Figure 3-2. Bridge assay setup showing location of nest cell, bridge, and food sources. A
dead adult cockroach was placed into the Fluon coated cup every other day.
The nest cell held 12 queens, 200 mg brood, and 500 nesting workers. 500
foraging workers were placed in the center of the arena.


50 mm


Treated
bridge

297 mm


56 cm































Figure 3-3. Glass bridge assay setup showing ants trailing along bridge.














CHAPTER 4
RESULTS

Repellency Assay

In all treatments, many of the ants immediately left the release dish and began to

explore the other dishes. Ants were observed walking on all dishes in all treatment

groups. There was no significant difference among the distribution of live ants in the

untreated and treated dishes for either the fipronil or the lambda-cyhalothrin treatments

throughout the duration of the repellency assay (Table 4-1). There were a large number

of dead ants in the lambda-cyhalothrin treated dish at 30, 45, and 60 min (Table 4-1).

Results from the t-test indicated that significantly fewer than 50% of responding ants

were in the fipronil treated dish at 15 min (t value = -2.69), indicating slight initial

repellency. Fipronil was not repellent at 30, 45, or 60 min. Results from the t-test

indicate that lambda-cyhalothrin was not repellent to worker pharaoh ants at any time

after treatment (Table 4-2).

Lethal Time Assay

In all treatments, worker ants clustered together in small groups after placement on

the treatment. On the glass surface workers began to exhibit grooming behavior,

including antennal and leg cleansing at -10 min in the lambda-cyhalothrin group and

after -30 min in the fipronil group. When pharaoh ant workers were exposed to glass

and wood treated with lambda-cyhalothrin, KD50 values for both glass and wood surfaces

were <1 h. Fipronil treatment caused significantly higher KD50 values than lambda-

cyhalothrin on glass and wood surfaces. Fipronil applied to wood was the only treatment









that caused delayed knockdown. Wood treated with fipronil had a KD50 value of >24 h

(1,950.00 min).

LT50 values for pharaoh ant workers on lambda-cyhalothrin ranged from 2 to 3 h on

glass and wood surfaces respectively (Table 4-4). When ants were exposed to fipronil,

the LT50 value was 496.54 min (8.3 h) on glass, and almost five times that on wood (39.9

h). A delay in mortality for >24 h only occurred in the fipronil treatment on the wood

surface. There was no worker mortality on water treated surfaces in the lethal time assay.

LT50 values for pharaoh ant queens were only slightly higher than those of workers

when exposed to lambda-cyhalothrin on both glass and wood (Table 4-4). There was no

significant difference in LT50 values between pharaoh ant workers and queens exposed to

lambda-cyhalothrin on wood. LT50 values for pharaoh ant queens exposed to fipronil on

glass were only slightly higher than LT50 values for workers (521.93 min for queens and

496.54 min for workers). On the wood surface, however, LT50 values for queens were

much higher than for workers (4,422.00 min for queens vs. 2,396.00 min for workers).

There was no queen mortality in the control lethal time assay.

Bridge Assay

After placement of the cockroach as food, ants were seen foraging across the bridge

within minutes. Within 30 min, a distinct trail of ants could be seen in most replicates.

In the glass bridge assay, ants were quickly knocked down and began to die within 1 h in

the treatment groups. Over 50% of the worker population was dead at 1 d for both of the

treatments (62.13% for fipronil and 54.90% for lambda-cyhalothrin). The control setup

never exceeded 50% mortality for the worker population for the duration of the test.

Nearly 80% of the worker population was dead at 14 d in the fipronil treated glass setup.

Similarly, the lambda-cyhalothrin treated glass setup resulted in 75% worker mortality









after 21 d (Table 4-5). Mortality for the water treated glass bridge was significantly less

than both the fipronil and lambda-cyhalothrin groups at time 1 h and remained so

throughout the duration of the experiment.

Worker mortality on the wood surface exhibited a delay in comparison to the glass

surface. Mortality was significantly higher in the lambda-cyhalothrin treatment (28.83

%) than either the control (13.37 %) or fipronil (15.88 %) treatment at 1 d. Mortality

after 1 d increased only slightly in the lambda-cyhalothrin treatment. The fipronil

treatment resulted in significantly higher mortality (40.38 %) than lambda-cyhalothrin

(31.20%) and the control (20.25 %) at 3 d and remained higher thereafter. At 14 and 21

d, the lambda-cyhalothrin group was not significantly different than the control group

(Table 4-5). The fipronil treatment resulted in 100% mortality after 21 d.

Queens generally remained inside the nest cells for the duration of the experiment.

However, in the glass bridge setups, a few queens were seen foraging at the 14 d mark in

two replicates of the fipronil treatment. In the glass bridge setup, no significant queen

mortality was observed throughout the duration of the experiment (Table 4-6). In the

wood setup, however, the fipronil treatment exhibited significantly greater queen

mortality at 14 d (59.73%) and 21 d (77.78%).

The mass of brood (by weight) decreased for fipronil and lambda-cyhalothrin

treatments. Fipronil treated glass resulted in a 43.55% reduction in brood mass (Table 4-

7). Lambda-cyhalothrin resulted in a 52.28% reduction in brood mass. The mass of

brood in the fipronil and lambda-cyhalothrin treatments were not significantly different at

28 d after placement of the treated bridges. In the wood bridge experiment, fipronil

exhibited a 79.20% reduction in mass of brood at the end of the experiment. The









Lambda-cyhalothrin also exhibited a significant difference in mass of brood compared to

the control 28 d after placement of the treated bridges (Table 4-7). However, this

reduction was small in comparison to the reduction in brood in the fipronil treatments

(17.19% for lambda-cyhalothrin).

The photographs of two of the nest cells from each treatment for the wood bridge

assay can be seen in Figs. 4-1 through 4-6. These present a representation of each of the

treatment groups. No visible difference was observed in the control replicates throughout

the duration of the experiment. A visible reduction of activity was observed in the

lambda-cyhalothrin replicate 4 (see Fig. 4-4). However, queens and brood are still visible

21 d after placement of the treated bridge. In the fipronil replicates, there is a strong

reduction in worker and queen number, as well as a reduction in brood. After 21 d, there

are very few queens and brood remaining (Figs. 4-5 and 4-6).












Table 4-1. Distribution of 50 worker ants in release, untreated, and water, fipronil, or lambda-cyhalothrin treated Petri dishes
Number of ants (mean SE) at time (min)
15 30
Treatment Dish Live Dead Live Dead
Control Water-treated 10.00 + 1.53a 0.00 + 0.00 9.33 0.67a 0.00 + 0.00a
Untreated 8.17 + 1.42a 0.00 + 0.00 10.67 0.56a 0.00 + 0.00a
Release 31.83 + 1.33b 0.00 + 0.00 30.00 + 1.15b 0.00 + 0.00a
Fipronil Treated 6.33 1.26a 0.00 + 0.00 9.83 + 1.62a 0.00 + 0.00a
Untreated 10.00 + 1.06a 0.00 + 0.00 11.17 2.06a 0.00 + 0.00a
Release 33.67 1.82b 0.00 + 0.00 29.00 1.71b 0.00 + 0.00a
X-cyhalothrin Treated 5.67 + 1.28a 0.00 + 0.00 6.50 + 1.34a 13.00 3.46b
Untreated 6.00 + 1.59a 0.00 + 0.00 6.33 + 1.65a 0.00 + 0.00a
Release 38.33 + 1.23b 0.00 + 0.00 24.17 + 1.87b 0.00 + 0.00a
Number of ants (mean SE) at time (min)
45 60
Control Water-treated 9.50 + 0.76a 0.00 + 0.OOa 9.50 + 0.76a 0.00 + 0.00a
Untreated 9.17 + 0.54a 0.00 + 0.OOa 8.83 + 0.60a 0.00 + 0.OOa
Release 31.33 + 0.88b 0.00 + 0.OOa 31.67 + 0.95b 0.00 + 0.OOa
Fipronil Treated 9.33 1.52a 0.00 + 0.OOa 11.00 + 0.97a 0.00 + 0.OOa
Untreated 9.67 + 1.33a 0.00 + 0.OOa 10.33 + 0.99a 0.00 + 0.OOa
Release 31.00 + 1.69b 0.00 + 0.OOa 28.67 + 1.41b 0.00 + 0.OOa
X-cyhalothrin Treated 4.33 0.56a 18.50 + 6.02b 3.50 + 0.50a 23.67 + 4.03b
Untreated 4.67 0.76a 0.00 + 0.OOa 2.17 + 0.48a 0.00 + 0.OOa
Release 22.50 + 1.28b 0.00 + 0.0a 20.67 + 1.74b 0.00 + 0.OOa

Means followed by the same letter within the same column treatment group are not significantly different
(p=0.05, Student Newman-Keuls test, SAS Institute, 2002).












Table 4-2. Repellency of fipronil or lambda-cyhalothrin treated glass to pharaoh ant workers


Time (min) Treatment n % Responding T statistic P
in Treated dish


15 Fipronil 6 38.76 2.69 0.04

X -cyhalothrin 6 48.59 -0.05 0.96

30 Fipronil 6 46.81 0.39 0.71

S-cyhalothrin 6 50.66 -0.21 0.83

45 Fipronil 6 49.11 0.16 0.88

S-cyhalothrin 6 48.11 0.50 0.64

60 Fipronil 6 51.57 -0.49 0.64

S-cyhalothrin 6 61.73 -1.52 0.19


Ha: Proportion of ants in treated dish 50% 0; One tailed t-test (SAS Institute, 2002)












Table 4-3. KD values of pharaoh ant workers when exposed to glass or wood treated with fipronil or lambda-cyhalothrin


Treatment Surface na


Fipronil


Slope


Glass 1,680 3.08 0.20


Wood 1,680 4.64 0.25



X-cyhalothrin Glass 720 4.82 + 0.30

Wood 1,440 1.96 0.14


KD50 min (95% CI)



194.98 (182.15-211.58)

1,950.00 (1,887.00-2,019.00)



19.07(18.11-20.17)

46.72 (42.23-52.79)


KD90 min (95% CI)



508.71 (432.51-625.77)

3,685.00 (3,408.00-4,055.00)



35.20 (32.11-39.43)

210.34 (161.81-297.11)


aTotal number of trials with 240 ants per trial
(Probit [SAS Institute 2002]).


X2



6.37

4.56



0.186

3.44


P



0.27

0.47



0.67

0.49












Table 4-4. LT values of pharaoh ant workers and queens when exposed to glass or wood treated with fipronil or lambda-cyhalothrin


Caste
Treatment Surface

Worker
Fipronil Glass
Wood

X-cyhalothrin Glass

Wood


Queen
Fipronil Glass

Wood

X-cyhalothrin Glass

Wood


nra Slope SE


1,200
960

1,440

960



960

720

1,440

1,440


18.29 1.14
7.98 + 0.40

4.00 + 0.21

3.30 + 0.27



12.52 + 0.64

14.53 1.41

4.18 + 0.42

5.98 + 0.28


LT50 min (95% CI)


496.54 (491.47-501.40)
2,396.00 (2,324.00-2,471.00)

137.84 (131.67-144.92)

158.24 (149.25-167.94)



521.93 (512.98-531.05)

4,422.00 (4,343.00-4,520.00)

164.45 (148.15-191.26)

172.13 (167.12-177.16)


LT90 min (95% CI)


583.49 (572.92-596.88)
3,468.00 (3,321.00-3,648.00)

288.12 (261.42-324.18)

386.82 (336.85-466.66)



660.68 (642.96-682.15)

5,418.00 (5,185.00-5,770.00)

333.38 (268.61-459.48)

282.05 (269.33-297.55)


aTotal number of trials with 240 ants per trial
(Probit, SAS Institute 2002).


X2 P


5.14
4.57

3.85

0.17



3.58

0.13

7.05

6.52


0.16
0.12

0.42

0.92



0.17

0.72

0.42

0.16











Table 4-5. Percent mortality of pharaoh ant workers in response to placement of glass or
wood bridges treated with water, fipronil, or lambda-cyhalothrin


Percent Mortality SE


Control

0.00 + 0.00a

8.78 + 0.871a

14.60 + 0.81a

21.27 + 0.62a

33.87 + 2.58a

43.07 1.35a


Wood h

Id

3

7

14


0.00 + 0.00a

13.37 + 0.63a

20.25 1.07a

38.65 1.30a

52.87 1.24a

57.30 + 0.03a


Fipronil

40.18 + 5.29b


62.13

68.70 +

72.70 +

79.90

86.90


11.32

15.88 +

40.38

60.48

84.25


5.07b

0.55b

0.54b

1.62b

1.10b


0.30b

0.96a

1.95b

0.90b

3.73b


100.00 3.50b


X-cyhalothrin

36.92 5.65b

54.90 4.60b

59.38 + 0.62b

62.12 + 0.19b

68.42 + 0.81b

75.73 + 0.33b


10.03 + 0.23b

28.83 + 0.56b

31.20 + 0.72c

45.50 + 0.69b

56.98 1.73a

63.13 + 1.15a


a Number of replicates with 1000 worker ants per replicate
Treatments applied at label rates for ant control; fipronil 0.06%, and lambda-cyhalothrin
0.015%
Means within a row followed by the same letter are not significantly different
(P=0.05, Student Newman-Keuls test, SAS Institute 2002).


Surface

Glass


Time

Ih

Id

3

7

14

21









Table 4-6. Percent mortality of pharaoh ant queens in response to placement of glass or
wood bridges treated with water, fipronil, or lambda-cyhalothrin


Percent Mortality SE


Surface

Glass


Wood 1 h

Id

3

7

14

21


Time

Ih

Id

3

7

14

21


Control

0.00 + 0.00

0.00 + 0.00

4.00 1.79

5.33 + 1.69

6.83 + 2.59

6.83 + 2.59



0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

1.38 1.38a

1.38 1.38a


aNumber of replicates with 12 queens per replicate
Treatments applied at label rates for ant control; fipronil 0.06%, and lambda-cyhalothrin
0.015%
Means within a row followed by the same letter are not significantly different
(P=0.05, Student Newman-Keuls test, SAS Institute 2002).


Fipronil

2.83 + 2.83

2.83 + 2.83

5.50 + 5.50

7.00 + 7.00

13.83 + 12.30

16.50 13.40



0.00 + 0.00

0.00 + 0.00

1.38 1.38

6.93 + 3.98

47.20 11.73b

77.73 10.02b


X-cyhalothrin

0.00 + 0.00

0.00 + 0.00

1.33 1.33

1.33 1.33

1.33 + 1.33

1.33 + 1.33



0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

0.00 + 0.00

2.77 1.75a

11.08 + 4.65a












Table 4-7. Mass of brood before and after placement of bridges treated with fipronil or lambda-cyhalothrin

Brood mass (mg SE)
Surface Time (d) n Control Fipronil X-cyhalothrin


200.33 + 0.16

194.68 12.80a

200.52 + 0.18

214.90 16.93a


200.00 + 0.21

112.90 10.82b

200.12 + 0.32

41.63 + 11.49b


200.10 + 0.15

95.48 12.54b

200.30 + 0.19

165.57 17.31c


Means within a row followed by the same letter are not significantly different
(P=0.05, Student Newman-Keuls test, (Institute 2002).


Glass



Wood





































I, .............. i:" i..... i... ................ D
Fig. 4-1. Series of photographs of Control (replicate 1) showing nest cell over time. A) 1
d before placement of bridge, B) 7 d after placement of bridge, C) 14 d after
placement of bridge, D) 21 d after placement of bridge. Notice there is not a
visible difference in activity from photograph A.
































'*' ( c- ----- D
Fig. 4-2. Series of photographs of Control (replicate 2) showing nest cell over time. A) 1
d before placement of bridge, B) 7 d after placement of bridge, C) 14 d after
placement of bridge, D) 21 d after placement of bridge. Notice there is not a
visible difference in activity, brood, or number of queens from photograph A.


Y

~~I





































C D
Fig 4-3. Series of photographs of lambda-cyhalothrin (replicate 1) showing the nest cell
over time. A) 1 d before placement of treated bridge, B) 7 d after placement of
treated bridge, C) 14 d after placement of treated bridge. Notice a slight
reduction in activity and brood, D) 21 d after placement of treated bridge. Notice
there is a slight increase in activity from photograph C, and there is not a large
difference in activity, brood, or queen number from photograph A.






































Fig 4-4. Series of photographs of lambda-cyhalothrin (replicate 2) showing nest cell over
time. A) 1 d before placement of treated bridge, B) 7 d after placement of treated
bridge, C) 14 d after placement of treated bridge. Notice a reduction in activity
and brood, D) 21 d after placement of treated bridge. Notice an increase in
activity and brood from that of photograph C. Also notice there is no queen
mortality.





















.. ...:...i,.. iiii



a a













S C D
Fig. 4-5. Series of photographs of fipronil (replicate 4) showing nest cell over time. A) 1
d before placement of treated bridge, B) 7 d after placement of treated bridge.
Notice a reduction in activity and several dead queens, C) 14 d after placement of
treated bridge. Notice a reduction in activity, brood, and dead queens, D) 21 d
after placement of treated bridge. Notice a reduction in activity, brood, and only
2 remaining queens.









.~v.


C D
Fig. 4-6. Series of photographs of fipronil (replicate 5) showing nest cell over time. A) 1
d before placement of treated bridge, B) 7 d after placement of treated bridge.
Notice a reduction in activity and a few dead queens, C) 14 d after placement of
treated bridge. Notice several dead queens, D) 21 d after placement of treated
bridge. Notice the reduction in brood and lack of queens.














CHAPTER 5
DISCUSSION

Foreword

Pharaoh ants are social insects that can be extremely difficult to control. They

exhibit many behaviors common to social insects including division of labor, trail

following and recruitment, and communication. These characteristics make control very

difficult. A small proportion of workers serve as foragers (Williams 1990, Vail 1996).

Treatments that aim to merely kill foraging workers will not be effective in eliminating

colonies. Additionally, pharaoh ants nest indoors, further hindering control efforts.

Treatments useful in excluding perimeter-invading ants may only cause trapping and

budding of pharaoh ant colonies inside (Vail 1996, Buczkowski et al. 2005).

Tests were performed to determine repellency, speed of kill, and the effects of a

residual treatment against colonies of the pharaoh ant. For a residual treatment to be an

effective control measure for pharaoh ants, the insecticide should mimic the

characteristics of effective baits. Effective baits should not cause foraging ants to avoid

the treatment, they should exhibit a delay in mortality of >24 h, and they should be

transferable from foraging workers to the rest of the colony.

For a residual treatment to be effective, the foraging worker ant must be willing to

walk onto a surface treated with the insecticide. An effective residual treatment for

control of pharaoh ant infestations should not only be nonrepellent, but the insecticide

should also exhibit a delay in mortality. When toxicants are encountered in smaller

concentrations, foraging ants have a longer period of time to transverse the treatment and









return to the nest. Delay in knockdown is also critical for spread of toxicant throughout

the population. If the ants are moribund, they will not be as effective in transferring

toxicant.

Repellency Assay

In this study, both fipronil and lambda-cyhalothrin treatments were not avoided by

worker ant movements. The lack of repellency of fipronil is in agreeance with many

studies on the nonrepellent action of fipronil to insects (Osbrink and Lax 2002, Ibrahim et

al. 2003, Scharf et al. 2004, Soeprono and Rust 2004b, Buczkowski et al. 2005, Hu

2005). The lack of repellency of lambda-cyhalothrin to pharaoh ant workers is consistent

with observations in other studies concerning the lack of repellency of ants to certain

pyrethroids. Pranschke et al. (2003) observed that red imported fire ants were not

repelled by bifenthrin. Consequently, treatments of fipronil and lambda-cyhalothrin

would not be avoided by worker ants and would cause worker ant mortality when applied

to surfaces in and around homes and structures.

It can be difficult to determine repellency of insecticides to ants. Many behavioral

aspects of pharaoh ants as well as characteristics of the insecticides can have drastic

influences on the interpretation of repellency. Such factors include the speed of kill of

the insecticides, nest relocation by the ants, formulation of the insecticides, and cessation

of trailing and recruitment.

The speed of kill of insecticides can affect the interpretation of repellency of the

chemical. Fast-acting insecticides, such as pyrethroids, cause the insects to die very

quickly and confound the data. Many of the ants will die on the treated surface, giving

the notion that there is a preference for the treated surface (Table 4-1). Our analysis was

modified to account for mortality when considering repellency. The distribution of live









ants was equal for treated and untreated dishes so the treatments were interpreted as being

nonrepellent.

Pharaoh ants can be easily induced to relocate their nests. Buczkowski et al. (2005)

found that pharaoh ants generally failed to relocate their nests under tile treated with

cypermethrin or bifenthrin. This was attributed to moderate repellency. However, more

information is necessary to determine if this is due to preferential nesting locations.

Buczkowski et al. (2005) also observed that pharaoh ants relocated their nesting location

35 d after exposure to cypermethrin. This may not necessarily be due to repellency of the

compound, however, since many factors can lead to nest abandonment (Holldobler and

Wilson 1990, Harris 1991, Buczkowski et al. 2005). My studies did not evaluate fipronil

or lambda-cyhalothrin for nest abandonment or nest relocation.

The formulation of an insecticide can have a strong influence on its degree of

repellency to ants (Knight and Rust 1990). Wettable powders were ranked as the most

repellent formulation to Argentine ants, followed by emulsifiable concentrates, and

granules (Knight and Rust 1990). In my studies, the lambda-cyhalothrin (Demand CS)

used was a microencapsulated formulation. The microencapsulation is thought to protect

the pyrethroid from environmental degradation. It may also serve to render the

pyrethroid nonrepellent. However, more research needs to be conducted on different

formulations of lambda-cyhalothrin to determine if this is true.

The cessation of trailing and recruitment behavior can give a false sense of

repellency. Soeprono and Rust (2004) suggested that pyrethroids do not repel Argentine

ants, but merely inhibit their ability to form recruitment trails. My results from the

repellency assay demonstrate that lambda-cyhalothrin exhibits no repellency to foraging









pharaoh ant workers for up to 60 min (t value = 1.52 at 60 min). Pharaoh ants were seen

immediately vacating the release dish and exploring both the treated as well as the

untreated dish. No food was provided in either dish and thus recruitment trails were not

formed.

Lethal Time Assay

Insecticides can have varying degrees of speed of kill. Fast-acting insecticides can

be effective in killing foraging ants and excluding ants from structures. Slow-acting

insecticides are more effective in eliminating colonies. Pharaoh ant workers were

knocked down very quickly when exposed to glass or wood treated with lambda-

cyhalothrin (KD5o values = 19.07 and 46.72 min, respectively). Although the ants were

quickly knocked down, it took several hours for death to occur. This was evident in both

the glass and wood treated lambda-cyhalothrin assays (LT5o values = 137.84 and 158.24

min, respectively). These results agree with previous studies with Argentine and red

imported fire ants, where pyrethroids act very quickly (Pranschke et al. 2003, Soeprono

and Rust 2004b). Smaller KD and LT values in the glass surface treatments were

probably due to the greater availability of the pyrethroid on the treated glass surface

(Chadwick 1985). Such small KD values indicate that foraging ants would not have

enough time to return to the nest and effectively spread the toxicant, especially since they

have been observed foraging for up to 45 m from the nest (Vail 1996).

Fipronil, when applied to glass, exhibited a longer knock down time for pharaoh

ant workers (KD5o value = 194.98 min) and longer lethal time (LT5o value = 496.54 min)

than lambda-cyhalothrin. When fipronil was applied to wood, however, there was a

drastic increase in lethal time values (LT5o value = 2396.00 min). My results show that

only fipronil treated wood exhibited a delay in mortality >24 h. This is consistent with









the speed of kill for effective bait toxicants, which exhibit a delay in mortality equivalent

to 1-4 days (Klotz et al. 1996, Klotz et al. 1997a, b, Rust et al. 2004).

Although queens do not perform extensive foraging duties, they often accompany

nest relocation events (Sudd 1960). Queens exposed to lambda-cyhalothrin treated glass

and wood exhibited a similar time to death than workers (LT5o values = 164.45 min and

172.13, respectively). This suggests that queens may not require larger doses of lambda-

cyhalothrin than workers for mortality. Queens exposed to fipronil treated glass also did

not exhibit a strong difference in lethal time values than workers (LT5o values = 521.93

for queens and 496.54 min for workers). However, queens exposed to fipronil treated

wood exhibited a much larger lethal time value than workers. This suggests that queens

may take much longer to die when exposed to lower doses of fipronil.

Bridge Assay

When exposed to a residual treatment, foraging worker ants are the first members

of the colony to be affected. Foraging workers contact the insecticide and return to the

nest. Nesting workers are the second members of the colony to be affected by a residual

treatment. If the foraging workers have enough time to get back to the nest they may

contaminate other workers by nest mate interactions, including grooming, trophallaxis, or

other social behaviors. Since the amount of insecticide is diluted when passed from ant

to ant, the nesting ants may require several doses from multiple foraging ants. If foraging

ants have the ability to cross the treatment and return to the nest several times, there

would be a greater transferability of the insecticide. A slow-acting insecticide would

allow the foraging workers enough time to cross the treatment several times without

becoming moribund.









Results from the glass bridge assay show that lambda-cyhalothrin treated glass will

successfully kill a large portion of the workers very quickly. This can be attributed to the

very fast knockdown and kill of workers. Over half of the worker population was killed

in 1 d. These affected foragers died before being able to spread the toxicant to the nest

cell. Consequently, -20% of the nesting worker ants lived through the duration of the

test. Lambda-cyhalothrin, when applied to nonporous surfaces such as glass can be

effective in quickly killing foraging workers, but should not be relied upon to eliminate

all the workers of a colony.

Results from the wood bridge assay showed that lambda-cyhalothrin treated wood

killed only a portion of the workers. After 14 d the pyrethroid produced no significant

difference in mortality than the control. This is consistent with James et al. (1998) who

observed that bands of 0.6% lambda-cyhalothrin applied to tree trunks were ineffective in

excluding Argentine ants after 5 wk. When lambda-cyhalothrin was applied to a porous

surface, it lost efficacy against foraging workers. Many of the nesting workers remained

unaffected throughout the duration of the test, as evidenced by the lack of high nesting

worker mortality.

Results from the fipronil treated glass bridge experiment showed that a large

portion of the worker population was killed over time. Over 80% of the worker

population was killed after 21 d. An 80% reduction in foraging ant has been interpreted

as providing satisfactory control in field experiments (Rust et al. 1996, Vega and Rust

2003). However, in my laboratory experiment, this reduction in worker population was

not sufficient to eliminate the colony.









Results from the fipronil treated wood bridge assay showed that when the toxicant

exhibited a delay in toxicity >24 h, there was enough time for the toxicant to spread from

ant to ant. Fipronil treated wood was also the only treatment to successfully eliminate all

worker ants. Lambda-cyhalothrin was found to be less effective after 14 d in the wood

bridge assay. Fipronil, however, was still effective in killing foraging workers through

the duration of the test.

In order to reach colony elimination, reproductive should be killed. In order to

affect reproductive located in the nest cells, the reproductive must encounter

insecticide-contaminated ants. Foraging ants must be able to transfer insecticide to

nesting ants, which then transfer insecticide to the reproductive. Both lambda-

cyhalothrin and fipronil, when applied to glass were ineffective in producing queen

mortality. This can be attributed to rapid speed of kill. When workers were exposed to

treatments on glass, lethal time values were all <24 h; lambda-cyhalothrin also was

ineffective in producing queen mortality due to rapid speed of kill (worker mortality

within a few hours). This rapid mortality did not allow sufficient time for workers to

contaminate the nest cell. The only treatment to exhibit significant queen mortality was

wood treated with fipronil. Wood treated with fipronil was also the only treatment to

effectively eliminate some of the colonies. This was also the only treatment to exhibit a

delay in mortality >24 h. In order to effectively transfer insecticide from worker ants to

queens, the insecticide must be slow-acting.

Pharaoh ants can start new colonies with just a small amount of brood (Vail 1996).

Brood must be affected in order to accomplish colony elimination. Brood can be affected

by residual treatments in several ways. The lowered mass of brood at the end of the









study was probably due to the lack of protein that was brought back to the nest, and

subsequent larval cannibalism (Holldobler and Wilson 1990). The mass of brood at the

end of the study for the lambda-cyhalothrin treated wood was significantly less than that

of the control. However, after the 14-day period, the mass of brood increased, suggesting

that the pyrethroid was no longer effective after 14 d. The foraging workers were able to

transverse the treatment, and gather enough protein to ensure larval survival and

production. This was also evident in the extreme reduction in the mass of brood at the

end of the experiment

Summary

Both fipronil and lambda-cyhalothrin effectively killed pharaoh ant workers and

queens when they are exposed to the treatment. No degree of repellency was found for

either insecticide. Fipronil and lambda-cyhalothrin exhibited varying times to death of

both workers and queens. There was however, an enormous difference in effects of a

residual treatment against colonies. Lambda-cyhalothrin was effective in killing a large

portion of workers, but did not eliminate the colony. Fipronil treated wood was the only

treatment to result in significant queen mortality and a large reduction in brood mass.

Some of the colonies in the fipronil applied to wood treatment were eliminated. Fipronil

applied to absorbent surfaces, such as wood shows the greatest potential for management

of pharaoh ant infestations.














APPENDIX
MANAGEMENT OF INSECT COLONIES

Pharaoh ant colonies were reared at the University of Florida (Gainesville, FL) in a

rearing room maintained at 27 1.50C, 40 7% RH. A space heater was operated

constantly to maintain a fairly constant temperature. Colonies were housed in plastic

trays (56 by 44 by 12 cm; Panel Controls, Greenville, SC) coated on the inner walls with

Fluon (AGC Chemicals, Bayonne, NJ) to prevent the ants from escaping. A sheet of

clear plastic (60 x 48 cm) was used to cover the trays and maintain a fairly constant

relative humidity inside the tray. Four to six nest cells were placed inside the tray to hold

queens, brood, and nesting workers. Cells consisted of square plaster-filled (Dentsply,

York, PA) plastic Petri dishes (100 x 15mm; Becton Dickinson Labware, Franklin Lakes,

NJ) (Williams 1990). A soldering iron was used to melt a 3 mm hole in the center of

each side of the bottom dish to allow the ants access. Yellow acetate paper covered the

nest cell to filter light and make it more attractive for brood rearing.

Colonies were provided ad libitum with water and 10% sugar water in

polypropylene round bottom centrifuge tubes (32 by 164 mm; Fisher Scientific,

Pittsburgh, PA) plugged with cotton balls. Freshly killed American cockroaches

(Periplaneta americana), obtained from the Urban Entomology laboratory, University of

Florida, Gainesville, FL were provided in paper cups three times per week. This

provided protein for oogenesis and larval development (Chapman 1998). When a new

cup of cockroaches was provided, the old cup was shaken to remove foraging ants, and

placed into a sealed bag. The bag was frozen for several days before disposal.

45






46


Dead ants were removed once per week with an index card. The bottom of the tray

was wiped with moist cotton balls to remove excrement and regurgitated food matter

once per week. All nest cells were transferred to a clean, Fluon coated tray once every

three months. Ants dispersed in the bottom of the old tray were collected on an index

card and transferred to the clean tray.
















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BIOGRAPHICAL SKETCH

David Melius was born on February 12, 1982, in Kingston, NY, to Steven and

Donna Melius. He has two sisters, Maria and Gina Melius. He and his family spent 15

years in Hurley, NY. In the summer of 1995, his father was transferred to Boca Raton,

FL. David attended Olympic Heights High School from 1995-2000. After high school,

David attended the University of Florida starting in August of 2000. He earned a

Bachelor of Science degree in entomology and nematology in December of 2003.

While earning his Bachelor of Science degree, David began working as a

laboratory technician for Dr. Phil Koehler in October of 2002. He gained experience

with rearing urban pest insects and managing insecticide field research. Such experience

captured his interest in graduate school and urban pest insects. He remained at the

University of Florida where he completed a master's degree in urban entomology.